Professor Hershel LaytonThe protagonist of the Professor Layton series, and one of the protagonists of this game. Layton is a world-renowned archaeology professor and puzzle-solver who always acts the role of a perfect gentleman.
Luke TritonLayton's young apprentice, who strives to become a proper English gentleman like the professor. He has the ability to speak to animals.
Phoenix WrightThe protagonist of the Ace Attorney series (well, most of it), and one of the protagonists of this game. Phoenix is a quick-thinking, honest defense attorney who is known for his ability to turn even the most hopeless of trials around.
Maya FeyPhoenix's assistant, the younger sister of his deceased mentor Mia Fey. As a spirit medium in training, she's been known to call on her sister to help Phoenix out of a pinch.
Aria (Mahoney Catalucia)A mysterious blonde girl living in Labyrinth City who finds herself pursued by forces beyond her control and accused of witchcraft. She seeks out Layton and Phoenix alike to ask for their help.
Sir Jiiken BarnrodA renowned knight of Labyrinth City who has taken it upon himself to serve as prosecutor of the city's witch trials.
Miss JordallaAria's teacher in London. In Labyrinth City, she acts as chief prosecutor and as the Story Teller's right-hand-woman.
The Story Teller (Literast Catalucia)The ruler of Labyrinth City; it is said that whatever he writes in his book becomes reality.
Johnny Smiles (Yutorino Gardner)A high-strung, highly-dedicated security guard working on a London barge, and a witness in the prologue case.
Olivia (Olive Ardente)Victim of the prologue case (though she survived), a chef working on the same barge as Gardner. She's actually a jewel smuggler who's been using the barge's Mr. Badger dolls as mules. When she saw Aria messing with one of the dolls with a jewel in it, she panicked and tried to shut her up permanently, but got clonked upside the head before she could finish the job.
Prosecutor BartleyThe prosecutor of the prologue case. Generally sort of wimpy.
NazomiThe librarian of Labyrinth City, who deeply loves puzzles. Any puzzles missed during the adventure will be sent to her, much like Granny Riddleton in the main Professor Layton games.
The Judge of Labyrinth CityA hooded, stern judge who presides over the Witch Trials. Though he bears a striking resemblance to his Ace Attorney counterpart, he's significantly more serious and level-headed.
KykeA head-in-the-clouds, unemployed old man who acts as a witness in Aria's trial. He also moonlights as a member of the local guard for the bell tower, and winds up testifying in Aria's second trial as well.
BerthaA down-to-earth, stubborn middle-aged woman who raises goats, and a witness in Aria's trial. The night of the crime, she took a bucket of milk from the scene that Aria had been carrying so it wouldn't go to waste, not knowing there was vital evidence at the bottom of it.
MadallaA young flower seller who acts as a witness in Aria's trial. She's the real killer of the bandits Alys and Terys; after hearing the Story Teller's stories, she became convinced that Aria was Bezella and decided to frame her for murder so the witch trials would come to an end.
KnightsAn unemployed young main so dedicated to becoming a knight that he'll only let himself be called, well, Knights. A witness in Aria's trial.
Emil SandbagA drunken middle-aged man who pops up time and again around Labyrinth City, and a witness in Aria's trial. Three months ago, he was also one of the people to find the alchemist Mist Belduke's body, and innocently took a bottle of deadly poison from the scene. When Layton was apparently turned to gold there in the present, he stole Layton's arm, sold it, and self-styled himself as "Emil Sandbag the Third" before being called to testify in Maya's trial.