The story begins with a car going at a high speed through a park. Inside, a man gives a young cloaked girla letter and tells her that should anything happen to him, to find the man written in the letter. Strange flying shapes with glowing eyes are seen whizzing past the car. The man notices them and calls them witches.
A one-eyed witch begins shooting beams of fire from it's golden hand at the car. They narrowly miss, and continue driving away from the witches. The witch from before then casts another spell, bringing two giant stone statues to life. Shocked, the man tries to avoid them, but fails and crashes into them. The girl wakes up and notices the man in pain on the grass. She holds him up, but he tells her to go without him, meanwhile, the witches start to fly around the two of them. Hearing police sirens, the girl runs off into the fog. Inspector Chelmey, Constable Barton and other policemen arrive on the scene. The Inspector looks up to see the man's car atop a tree.
Completely stunned, Chelmey says to himself that this looks to be a tough case, but that their's one man he knows who could get to the bottom of it. Meanwhile, inside London, Professor Layton tells Luke about how in the Middle Ages, people believed heavily in magic and would have "witch trials". A lightning flash is seen and it begins raining outside. Luke then asks the professor if witches really existed back then. Meanwhile, the cloaked girl was seen running down an alley holding a book. Layton tells Luke that witch trials certainly existed, but that nobody actually used magic, and that witches were nothing more that superstition and legend.
Mwanwhile, high above the skies of London a plane is seen.
Inside, there are two American passengers, Defence Attorney Phoenix Wright and his assistant, Spirit Medium Maya Fey. Maya is excited to see London and tries to get Phoenix to look. Wright asks Maya to let him rest and tries to get back to sleep. She says that Wright won't win in an English court with that attitude, though Wright states that because he's here on an exchange visit courtesy of the Legal League of Attorneys, he won't officially be defending anyone in court. Maya then pulls on the eye and mouth pieces that he's wearing and lets them go, causing Phoenix to jump in pain. Maya tells him that he always works better under pressure, but to not underestimate how the trial will go in England. Phoenix puts his earphones on and tells her not to talk like that, stating that she'll only jinx it. Below them, on the Tower Bridge, the one-eyed witch opens her wings and soars through the sky.
Back at Layton's office, Luke began complaining about the weather, citing that it was unusual for that time of year. Layton agreed, but noticed that it was beginning to ease up. To take his mind off the weather, Layton told Luke to examine the bookshelf. Luke then went over to the bookshelf and noticed a picture book. Surprised that he would have one he then noticed a puzzle inside the book. After Luke solved it Layton congratulated him and the pair prepared to head home. Just before they could leave they heard a knock at the door. Layton wondered who it could be at such a late hour, stating that he wasn't expecting anyone. Luke then answered the door, to see the young cloaked girl from before.
She asked for the professor, and he in return asked what he needed him for. She then gave him a letter and asked him to read it. He noticed that it was from his former student, detective Carmine Accidenti. The girl began to tell them about Carmine, but then stopped herself. Layton stated that letter must contain something important and began to read it.
In the letter, Carmine stated that his life was in danger and that he was being chased by something terrifying. He also mentioned a town called Labyrinthia and how it was not found on any map, telling them that he had seen many unbelievable things while he was there. Realising that the town held a mysterious secret he decided to uncover it, but was caught. He escaped the town with the young girl, called Espella, and said that their pursuers were actually after her, ending the note by asking the professor too help her.
Layton asks the girl if she's Espella, to which she says that she is. He then asks her what happened, but she is unsure herself and only remembers being chased, attacked and seeing Carmine injured. Espella then begs the professor to help him, but Layton reassures her that, knowing Carmine, he will be fine. He tells her that he's more concerned for Espella's safety and asks if she feels all right. She says she's alright though Layton insists that she take a little rest. Layton and Luke discuss the possibility that Espella's pursuers will come too the office and Luke then goes to lock the door.
The professor ask's Espella for a better summary of what happened, noting that without a clearer understanding they won't be able to protect her. Surprised that he wants to protect her, Layton tells her that it's the duty of any gentleman to protect a lady in need. Layton then asks who it actually is that is pursuing her. She responds by telling him that she's being chased by witches, shocking both the professor and Luke.
She tells them that they will be in danger if she stays here. Layton tells her not to worry and that they will guard her. He then asks her if she came from Labyrinthia, to which she bewilderingly said yes. Luke asked her what was wrong. Espella then asked if they weren't already in Labyrinthia, a confused Luke told her that they were in London, England, but she had never heard of the place before. Espella replied that she never came across the name in Labyrinthia. Layton then asked where Labyrinthia was, she told him that anything noteworthy about Labyrinthia could be found in the book she was holding. Layton asked her if he could read the book and she agreed.
Both Layton and Luke started reading it and both noticed that the words began the move on their own. Luke thought he was seeing things, but Layton confirmed that he saw the letters move too. Looking through the book, Luke saw a picture of Labyrinthia and noted that the buildings looked really old. Layton then added that he had never seen the town before. Layton told her that the book looked valuable, she told him that the book was called the Historia Labyrinthia and also told they that it contained the entire Story.
Layton suggests that the book must contain something about the witches. She says it does, and also that it contained information on all the events that had took place in Labyrinthia and on all people that living there. She states that anything written in the book will happen.
Luke talks to the professor about all that has been said and tells him that he finds it hard too believe. A pigeon then taps on the window. Luke goes to let the bird out of the rain, Espella quickly tells him not to but is too late. It begins to fly around the room and mysteriously turns the electricity off when it passes the light. A strong gust of wind then appears in the middle of the room, and appearing from the thick of it is the one-eyed witch from before.
It incants a spell that renders Espella unconscious and lifts her off her feet toward the witch and into her arms. Layton quickly tries too rush toward her but the witch blasts him a gust of wind, throwing books and artefacts around the room and keeping the professor at bay. Layton edges closer to the two but the witch soars past him and goes through the office window, destroying it. Luke asks Layton what that thing was, and as they both look out of their broken window, Layton tells him that that was a witch.
Luke worriedly exclaimed that Espella had been kidnapped, Layton was surprised that witch was able to get inside the office by using trickery but was ashamed that he had failed to protect her. Luke asks him if that really was a witch that used magic. Though unsure, he knew that something extraordinary took place. He then told Luke that he was going too look for Espella, Luke quickly interjected saying that he was going too. After a bit of consideration, Layton agreed and they both leave and head toward the River Thames.
After arriving near the Thames, they spot Inspector Chelmey and Constable Barton. Chelmey tells Barton that the case case stumped him with Barton adding that it's a total mystery. This angers Chelmey, who only wants answers not agreement. Layton and Luke see their heated discussion and wonders if it has anything to do with the witch that took Espella. Chelmey notices the two of them and begins too tell them to leave the area, until he notices that it's Layton and Luke.
Layton asks if something has happened, Chelmey replies that a motor vehicle had been heavily damaged. Layton wonders why a Scotland Yard inspector would investigate a car accident, and suggests that something unusual happened. Barton quickly says that it's the most unusual accident he's ever seen, angering Chelmey.
Chelmey then tells Layton to leave it to the police, but the professor tells that he may be able to help solve the case. Barton states that even they haven't found any clues yet, angering Chelmey again. The inspector tells Layton that if he wants to find clues he should go to the park, the scene of the crime, himself. And reminds him that if he finds anything, he should let him know as well. Luke theorises that the car accident may have something to do with their own investigation, the two then head to the park. Chelmey then says with the amount of cases that he's got he needs all the help he can get.
At the park, Luke is quick to notice a car on top of a tree, surprising both of them. Layton tells Luke that they'll need too investigate the car more closely. Luke notices that the car is seriously damaged and asks the professor if he thinks the witches are responsible. He replies that it's possible as the accident looks to have occurred only moments ago.
Layton then notices a mark on the bonnet of the car, but before he can investigate further, a constable intervenes and asks the two of them to leave. Luke tells them that Inspector Chelmey had given them permission. The constable then asked them who they were. Layton introduces himself which surprises the policeman. To make sure that he's the real professor, he gives Layton a puzzle. Layton solves it and the constable let's them investigate the area, and requests that he sign his helmet later on.
Layton asks Luke to check the mark on the car's bonnet, and he notices that it looks like a hand print. He also mentions that the hand print is too big to be made by a human. They investigate some more and find a stone statue, Luke notices it's open hand and wonders if it hit the car when it crashed. Layton states it as a possibility but notes that the hand print on the car is right handed. Seeing as the statue is holding a spear in it's right hand, Luke says that it's impossible the print came from the statue. Luke wondered if the statue had moved, Layton remained uncertain though was sure that it was connected to the one-eyed witch. Layton then tells Luke that they sgould now head back to Chelmey and report their findings to him, he also wants to ask who was in the car when it crashed. They both leave the park.
Layton returns to Chelmey and asks him who the car's driver was, Chelmey says that he wasn't seriously hurt and that he was preparing him for questioning. Layton then asked if the drivers name was Carmine Accidenti. Shocked that he knew Carmine, Chelmey confirmed that he was the driver. Luke then told the professor that that must have been the car that Carmine and Espella were attacked in, Layton agreed. He then asked the inspector if there was anything suggesting that their was anyone else in the car at the time, Chelmey told him that there wasn't.
Layton then asked if they're was anything unusual about the crash that he hadn't mentioned, though nothing came to his mind. Barton then mentioned how they had seen people wearing Halloween costumes around, angering Chelmey yet again. Layton inquired about them and was told that they were seen around Tower Bridge. Realising that they may have a lead, Luke thanked the inspector and head to Tower Bridge
Layton and Luke arrive at the landmark and notice that there are no civilians on the bridge. Luke blames the weather but the professor senses a sinister presence in the air. Luke asks him not to talk like that, but they are blinded by a bright accompanied by a loud noise. They then notice a figure in a purple cloak, with glowing red eyes floating further down the bridge. It quickly disappears, and they then travel to it's original location.
When they get there, Layton tells Luke that the dark presence he felt before is gone. They then find Espella's cloak on the ground, next to a strange marking on the bridge's wall. They decide to investigate the marking further, until Luke begins to hear something. He then notices that place is shaking and has dislodged some bricks from the marked wall. After fixing it, the wall opens revealing a staircase, which Luke then falls down. Layton is quick to rush to his apprentices aid, and ask him if he's alright. Luke says he is, but then notices that they are facing a long tunnel. Luke then remembers Espella, Layton tells him that they both need to be careful and they return to their search. Luke finds her, but notices that she's tied up, they then begin to untie her.
After she is free from her bindings, Espella thanks both Layton and Luke. The purple cloaked figure from before then appears from the other end of the tunnel and begins approaching the trio. Espella tells them to save themselves, but Layton tells her not to worry and that he has an idea. Layton and Espella run toward the other end of the bridge, but it rises due to a passing cruiser. Shadowy figures appear around them, and then the witch appears atop the raised bridge and demands that Layton hand Espella over. It then sucks the two closer to her which causes Espella's cloak to be blow away toward the sea. It was then revealed that it was actually Luke under cloak wearing some makeshift stilts, while also holding the Historia Labyrinthia.
The cloak lands on the railing of the passing cruiser. Espella then comes out from between two pieces of cargo and takes her cloak, she looks out toward Tower Bridge saying Layton's name. Back on the bridge Layton asked the witch who they were and why the were after Espella.
It laughed at him and called him a fool, while mentioning that they had unwittingly walked into the middle of the story. It said that they would see what it meant with there on eyes before vanishing.
They both pondered what she meant, then decided to look for Espella at the port the ship was heading to. The Historia Labyrinthia then begins to shake violently in Luke's hand and opens itself to a page showing Layton and Luke in Labyrinthia. A blue light envelops them and they disappear. The book then closes.