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Lucy BakerThe main protagonist and the narrator. She's a DC (detective constable) who works under Inspector Layton.
- Curtains Match the Window
- Declarative Finger: One of her frequent sprites is this.
- Expy: Of Luke Triton from the original Professor Layton series. They even wear similar-looking hats.
- Na´ve Newcomer: She's start out at the beginning of the game as a new officer starting out as Alfendi's assistant.
- Nice Hat
- Player Character: Despite the title Layton Brothers, she's the character you actually play as.
- Plucky Girl: Lampshaded by the "other" Alfendi in case 3.
- Pungeon Master: Has a tendency to be this. This is taken Up to Eleven in Case 8 however, as it dealt with animals and many of her responses were puns about the people/environment, especially during her interrogation with the suspects.
Alfendi LaytonThe titular protagonist although he's more of a deuteragonist. A brilliant inspector of Scotland Yard and the son of Professor Hershel Layton. He has another 'side' to him where his personality changes into a more threatening and criminal-obsessed one.
- Anime Hair
- Fake Memories: "Placid" Layton claims that he is the one who killed Keelan while "Potty" Layton has no memories beyond the shooting. The fake memories are courtesy of Justin Lawson.
- Exposition Fairy: He essentially serves as a guide teaching you how to play the game.
- Good Cop/Bad Cop
- Leeroy Jenkins: Averted. Alfendi is much more likely to think things over before jumping ahead, the only exception being case 9 where people's safety was at jeopardy if he didn't comply with Diane. However, the real Prof doesn't seem to be this, jumping ahead of his colleagues when Keelan Makepeace's location was first discovered, leading him to a trap .
- Jekyll & Hyde: Has another side which is more violent and even threatens to kill someone during interrgations.
- Mentor Archetype: To Lucy.
- Missing Mom: Which is the question of many fans.
- Nice Guy: Is generally level-headed and usually says things in the nicest way possible, despite some of the verbal insults some of the suspects give him. Until the other Alfendi comes out.
- The Professor: Serves as Lucy's mentor. Isn't too fond of the nick-name though.
- Tomato in the Mirror: His other rude personality is actually his original personality. The nice personality was a new personality that appeared after the incident where he was shot.
- Split Personality: Even his profile describes him as this.
- Super-Powered Evil Side: His other side often provides the key deduction that ultimately cracks the case due to it being able to think like a criminal.
The CommissionerThe... commissioner of Scotland Yard. His name is Barton. Yes, the same Barton who was a constable and worked under Inspector Chelmey.
- Adaptational Badass: Went from Chelmey's sidekick to commissioner of the Scotland Yard.
- Big Eater: In the ending credits, we see him eat a large plate of food.
- The Commissioner Gordon: Averted since the main characters are members of the police force. But played straight in the last scene of the game which is a Sequel Hook showing the Commisioner calling Amateur Sleuth Hershel Layton asking for help.
- Continuity Nod: He used to be Inspector Chelmey's sidekick.
- Cool Old Guy
Deputy Commissioner ChanThe Deputy Commissioner who talked with Lucy Baker in the prologue.
- Hypocrite: Somewhat of one when it comes to superstitions.
- Jerk with a Heart of Gold: Despite being incredibly discouraging to Lucy about working in the Mystery Room, he does worry about her safety. Mainly because the inspector she works under with has a split personality.
Florence SichA forensics analyst. She gets sick very often and as a result, goes around in an ambulance.
- Ill Girl: Is hooked up to an IV machine and sneezes quite a bit.
- Meaningful Name: Her surname is pronounced "sick".
- It also sounds very similar to "Forensic."
- Proper Tights With Skirt: Noticeable in her full profile picture.
Justin LawsonA burly Scotland Yard inspector and basically the polar opposite of Inspector Layton.
- Dirty Cop
- Hardboiled Detective
- Jerk Ass With A Heart Of Gold: Despite being responsible for at least half the Jigsaw Killings, he couldn't bring himself to kill Alfendi twice despite having the chance. He is also the one who suggested to listen to Alfendi's statements.
- Treacherous Advisor: While she does this to other people as well, Lucy generally confronts Alfendi's behaviour to Justin, who at one point said that if Alfendi mentions any specific people or names, that Lucy should secretly talk to Justin about it. Turns out he is the main reason why Alfendi has his split personality in the first place.
Dustin ScowersThe janitor who often picks up information about Scotland Yard during his duties.
'Sniffer' HagueA detective working under Justin Lawson. Apparently has a great sense of smell.
Hilda PertinaxAn Interpol agent working with Scotland Yard to find a murderer who stole an artifact.
- Dub Name Change: From Hilda Swanson.
- Lovable Alpha Bitch: Downplayed. She scolds Lucy for eavesdropping on her conversation with Alfendi despite it being quite loud, a bit bossy and was initially adamant of convicting Alfendi as the killer. However, she generally looks after Lucy, telling her Placid Prof was the fake persona and offering her comfort. By the end of case 9, she sides with Lucy on her theory about the true killer (though Hilda will begin to regret it if the player makes a mistake).
Sandy AldwichA woman found dead on her vacation at a five-star hotel. For some odd reason, her hand was found in her sandwich she ordered.
- Asshole Victim: She gets her money from her boyfriends and afterwards dump them for another man.
- Dying Clue: Supposedly left one as she was apparently a fan of crime fiction. Too bad the very existence of the dying message gave it away as a fake.
- Gold Digger: Possibly. According to the killer, they used to be an item until he ran out of money, at which point she promptly dumped him. Also, her current lover is a wealthy businessman who is paying for everything on their luxurious vacation.
- Punny Name: Sandwich.
Jack PotsbyA loser who recently won a huge sum of money.
- Punny Name: Jackpot.
Gloria BlaiseThe lead actor of a play where she was the heroine.
- Driven to Suicide: She made it elaborate enough to frame Strapping for murder.
- Punny Name: Blaze of glory.
Archie O'LogieA world-famous archaeology professor.
- Punny Name: Archaeology
Dwight DreadA DJ who hosted a famous radio show.
- Asshole Victim: Once he found out de Bonair had been stealing profits from the radio station, he blackmailed him by having de Bonair give him money.
- Meaningful Name: Dread refers to his dreadlocks of his wig.
Anice BrewerA tea lady working at a radio station.
- Fangirl: Of Dwight Dread. Unfortunately, this was exploited to help the murderer kill the main victim and then pin the blame on her
- Proper Tights With Skirt
- Punny Name: A nice brew
- Red Herring: In-universe thanks to Sniffer.
- Spanner in the Works: Miraculously, she survives her poisoning long enough to leave a Dying Clue.
- Unwitting Pawn
Severino AgonniA mafia boss and head of the Agonni family.
Barbarossa SassinaAgonni's right-hand man.
- Asshole Victim: Not according to Mistry's story of him.
- Dragon with an Agenda: Poisoned half of the members while he burned his boss.
- Punny Name: Barbarous assassin
Pearce 'Blood' BoyleThe Agonni family's strongman.
Angelo FassiA new recruit of the Agonni family and served as their pretty boy.
- Asshole Victim
- Bishounen (In-Universe): It is what his profile describes him as.
- Punny Name: Angel face
Keelan MakepeaceThe serial killer responsible for the Jigsaw Puzzle Killings.
- Asshole Victim
- Bad Guys Do the Dirty Work: Some of his killings were requested by Justin when the Yard couldn't catch the criminals themselves
- The Killer Becomes the Killed
- Posthumous Character: Died from the incident four years ago
- Punny Name: Kill and make peace.
- Asshole Victim: At least that's what his colleagues think of him as.
Diane MakepeaceDetailed more on the Suspects section.
Bosco FelpsAldwich's lover and for business purposes, refused to tell the police his identity.
- Spell My Name with an "S": An in-universe example. Carrière thought his last name was "Phelps" instead of Felps".
- No Celebrities Were Harmed: This guy is practically Mr. T in a business suit. One of his arrows even says "I pity the fool!" and Lucy warned him to "B-A-round!" for further questioning.
Zach CarrièreA bellboy who brought Aldwich her sandwich.
- Fanboy: He is a self-admitted fan of crime fiction.
- French Jerk: Double Subverted. He tries to be helpful to Alfendi and Lucy until he learns that he is their main suspect.
- Starter Villain
Vera WipovskyThe hotel cleaner who found Aldwich's dead body.
- Red Herring: If you think that her description gives her the possible motive of killing Aldwich because of her possibly cheating on her husband, then think again.
- Team Mom: Has this relationship with her hotel employees.
Goldie Potsby-MahnJack Potsby's wife.
- Black Widow: She had three husbands before Potsby and became rich from their life insurance. Despite that, she's apparently innocent according to the police and insurance companies. She messed up when it came to killing Potsby.
- Graceful Ladies Like Purple: Wears a slinky purple dress.
- Multi Coloured Hair: Her bangs are white, her side bangs are lavender and the rest of her hair is black.
- Pretty in Mink: Has a white fur coat◊.
Chase M. DownesThe muscular concierge of the apartment where Potsby lived in.
- Red Herring: Him being muscular and joining an exclusive gym has nothing to do with the case.
- Punny Name: Chase him down.
Buster NicksAn old serial burglar.
BingoPotsby-Mahn's new boyfriend.
- Beast Man: Looks less like a human and more like a lion standing on his hind legs. Goldie even keeps him on a leash like a pet cat!
- The Voiceless: Does speak sometimes but it's basically barking.
Roscoe StrappingA famous actor partly known for his debauchery.
- All Men Are Perverts: He not only hits on Lucy, he assumes that Alfendi is getting some action from her. Because he believes that's what men do when they have cute female assistants.
- Demoted to Extra: In-Universe. In the credits he's shown as a lowly stagehand while Knox and Clegg are on stage. Presumably, the results from the case created a scandal that ruined his acting career.
- Your Cheating Heart: Even though he's engaged to Blaise, he cheats on her with Knox, the supporting actor.
Destiny KnoxThe supporting actor for the play.
- Karma Houdini: By the looks of the credits, she doesn't get punished for lying about hearing Blaise scream.
- Punny Name: Destiny knocks (on your door)
Bray CleggA stagehand of the play.
- Ascended Fanboy: Went from stagehand to an actor as seen in the credits.
- Framing the Guilty Party: Strapping knocked him out to frame him, since he genuinely thought he switched the guns. He was wrong.
- Punny Name: Break a leg. Also possibly punning a kleig lighting on a stage.
Doug ScowersDustin Scowers's older brother and a student of O'Logie.
- Punny Name: Dig (and) scour. Another meaning of "scour" (apart from the one used for his brother) is to "seek" or "search", and he's an archaeologist.
Micah SasucasaA hotel owner.
- Bilingual Bonus: If you know Spanish, then try to say his name out loud.
Mariana EtistaA young woman working as a hotel employee. Actually a fake identity used by Diane Makepeace.
Chico CaretaA local of a village who volunteered to be a local guide for the archaeologists.
- Color Coded Emotions: He emotes through various colored masks. Red for Anger, Blue for Sadness/Nervousness, and Green for Joy/Laughter.
- Eloquent in My Native Tongue: Not only is his English is broken and peppered with Spanish words, but he is unable to read or write English as well. Justified in that he has only been learning English from Mariana for the past six months and is still learning.
- El Spanish O
- Love Martyr: He recognized Mariana's voice coming from the statue, which implies that he knew he was killing on her behalf, and not on that of a demon.
Dolly HollerdayA famous singer and a guest in Dread's show.
- The Ditz
- Giftedly Bad: She sees herself as a great singer but her profile claims she's horrible.
- Ms. Fanservice: Her profile says she's able to distract from her bad singing by acting sexy onstage.
- Red Herring: Initially, not knowing much about the case, Alfendi supposes on a less-than-50%-chance she's the killer.
- Shipper on Deck: For Lucy and Alfendi, it seems.
Melody SmithA talent agent who became Hollerday's manager.
Mike de BonairThe friendly director of the radio show.
- Deadpan Snarker: During his interrogation, he will make snide comments about the Metro's efficiency.
- Bitch in Sheep's Clothing: You see his true colours during the interrogation. He doesn't even know the name of the other co-worker he tricked and killed.
- Meaningful Name: "de Bonair" equals "debonair".
- Multi Coloured Hair: Has two white bands of hair in the side.
- Smug Snake: Especially in the final part of his interrogation.
Randal MannThe sound technician of the radio show.
Uttar MistryThe delivery boy of a Dale Herl's Deli, a delicatessen.
- Blush Sticker: When he fanboys about the second-in-command mafia murderer and victim.
- Children Are Innocent: When Alfendi tells Lucy he is 100% certain Mistry is not the culprit, Lucy accuses him of thinking that because he's only a kid.
- Punny Name: "Utter mystery". Not like you needed to ask.
- Loony Fan: For someone who was worried about being killed by the mafia agent, he seems to be big fan of him.
Diane MakepeaceThe daughter of Keelan Makepeace.
- Affably Evil: Lampshaded by Lucy in Case 9.
- Avenging the Villain: Her acts against Layton are out of avenging her father's death
- Bait-and-Switch Boss: Despite being the main villain of cases 4, 6, 7, and 8 which seeming makes her the final villain, she's killed offscreen at the start of the case 9 and confrontation against the final boss of the game is to uncover her killer.
- Cute and Psycho
- Daddy's Little Villain: Of Keelan Makepeace
- Elegant Gothic Lolita: Played even more straight since she is an adult.
- The Killer Becomes the Killed: By the same way her father died no less
- Little Miss Badass: Lampshaded in her profile that despite her innocent appearance, she has international notoriety.
- Nice Hat: As part of her gothic lolita outfit.
- Perky Goth: Which generally adds to her creepiness.
- Punny Name: Die and make peace.
- Revenge Before Reason
- Serial Killer
- Soft-Spoken Sadist
- You Killed My Father: She wants revenge against Alfendi Layton for killing her father.
CatThe wife of Pig.
- Gold Digger: Although her profile states that she wants to claim the life insurance policy on Pig, she insists she isn't one as she wouldn't get the money Pig passed down to her. She's not a gold digger as she didn't kill her husband.