Damsel Scrappy: Espella to some. She's the defendant in most of the game's trials and constantly needs people coming to her rescue. In fact, one of the first puzzles is untying her after she gets captured.
Ending Fatigue: The final trial spans three chapters, when every other trial only lasted one, and the third part basically consists of one Info Dump after another with only a few brief testimonies to break them up. The actual finale takes a while too.
First Installment Wins: In addition to using the classic variations of Objection and Cornered themes, Phoenix's lifebar during court segments takes the form of icons again which were only seen in the first game up until this point. Though this could make them easier to quantify as picarats.
Harsher in Hindsight: "Barnham's Wild Ride" from the bonus content. His horse accidentally killed the dog that startled it — Constantine's parent.
Les Yay: Lettie Mailer does seemingly have a huge crush on Jean, admitting she found Jean attractive while she was posing as a boy and even bringing her flowers in the ending. (In the special episodes, Lettie also says that she would've preferred it if Jean had posed as a male longer, which makes you wonder how she truly views her friend.)
Upon fully remembering their childhood friendship and what happened on the day of the Legendary Great Fire, Espella and Darklaw/Eve have fairly strong traces of this. Even beyond Eve rushing to the Bell Tower to prevent Espella from committing suicide by grabbing her hand at the risk of certain death for them both and then shielding Espella's body with her own as they fall, their apologies towards one another have an air of great awkwardness that could be interpreted as something more than two old friends making up for lost time. As Eve and Barnham have... something going on between them in the special episodes, Eve could be read as bisexual. Of course, this can all be chalked up to how shy Eve really is when she's not playing a role.
Layton gets turned into gold at the end of Chapter 3, and Maya is locked in a cage and plunged into the courtroom's fire at the end of Chapter 4. Even if you haven't seen the trailers and know there's still far more for them to do later in the game, it doesn't take a genius to figure out they're not gone for good.
As another example, like Layton would really suddenly turn traitor/villain and prosecute Espella during the last trial for no reason. It's presented as a "shocking twist" cutscene, but it makes sense in context and there are very good reasons for the scene progressing the way it does.
One during the finale: Espella's father reveals he's suffering from a fatal, incurable disease. After everything she went through, something like this would practically qualify as a Downer Ending, so it comes as no surprise that a cure has actually been found.
Flynch's unbelievably weak and frail "OBJECTION!" shout. Thankfully, it's short-lived, as he only appears in the tutorial case and is then never seen again.
"Barnham! Barnham! Barnham!" After a few repetitions of that chant, you may find yourself wishing you could strangle everyone in the viewing gallery.
Mailer's loud "SIIIIIIIIIIIIIIIIIIIIIIIIIIIIR!" Even in-universe, considered an annoyance by all. As a bonus, if Phoenix is the next character to speak (or think) afterwards, it'll be accompanied by a brief high-pitched beep to signify his ears are ringing. At one point, even Barnham and the Judge's ears are shown ringing.
Mary has an animation where she squeezes her goat hard, causing it to wail loudly for the entire duration of the dialogue box. Fortunately, the sound of the goat is not heard except during trials.
Birdly's tunes. Especially during the cross examination where he's Emeer's personal bard. They replace the music during the dialogue box and consist of a repetitive lute and bagpipe melody. There also exists an even more repetitive sad version of the song.
Barnham's clanky desk slamming. It's not that annoying at first, but expect to hear it A LOT throughout the trial scenes.
Narm: Some of the voice acting can fall into this. This also pertains to dramatic lines that get cut off by the text boxes.
A lot of Phoenix's lines are delivered very flatly, leading to many Dull Surprise moments from him. This is especially prevalent during emotional moments.
Narm Charm: In true Layton fashion, The Reveal is so utterly absurd in its efforts to create a "rational" explanation for the supernatural happenings—involving some hilariously made-up science, mass hypnosis, a staggeringly over-elaborate plot, no apparent consideration of trauma therapy, and the flawless cover up of the deaths of an ENTIRE village in the modern era— that you really have to salute the writers for coming up with it.
Pandering to the Base: The whole game is a present to people who enjoy both the Layton and the Ace Attorney series, but there is no reason to put Edgeworth in the post-credits scene other than to cause a few fangasms.
Player Punch: Once you find out what happens to those convicted of witchcraft, having to accuse anyone tends to come off as this.
Having to cross-examine a grieving Luke in the 3rd trial.
Layton and Maya's apparent deaths. Though it should be fairly obvious the narrative would never pull something like this for real (especially in a crossover game of all things), it still hits hard because you get to see the reactions of the characters closest to them as if they really had died. So even if you know that the game will think of some way to bring them back, you get a long and painful look at how the characters would react if this really were to happen.
Espella's attempted suicide towards the end of the final trial. It comes out of nowhere in the middle of a cross-examination with very little warning, and an unsuspecting player might feel like they're hit a particularly cruel Bad Ending. Nope, it's actually required to progress.
Surprise Difficulty: Ace Attorney fans who played Dual Destinies first are probably going to be taken off guard by how much harder this game's trials are, mostly because you need to start the cross examination from the beginning even if you save during it and there's no option to just restart from the same statement with full life if you run out.
That One Level: The band of vigilantes' testimony in the final trial, due to having so many similar characters, including some that serve next to no purpose beyond wasting your time.
That One Puzzle: Eccentric Tailor: it involves filling out a specific pattern with an U-shaped stamp that fills in any unfilled squares it stamps and erases any already filled squares. Even worse, the final DLC episode requires you to complete an even harder version of the puzzle.
Underused Game Mechanic: Puzzles with Contradictions. The concept in and of itself is an amazing new twist on the formulas of both original series, but it is only used once in the entire game, albeit at a critical point. Also, you are again able to present profiles in cross examinations, but it's never used.
The Woobie: Luke during the 3rd trial, having taken the stand due to extreme grief over Layton's apparent death. The game does a very good job of making you feel awful every time you have to press his testimony. Cross-examining a child isn't something that happens often in Ace Attorney games, but one of the few times it did at least the kid had some annoying qualities to balance it out. Not here.
Espella (depending on how annoying the individual player finds her), as she spends the entire game being accused, more than once, of being a witch, and to make matters worse even she starts thinking she's guilty, and it gets even worse than that after she (mistakenly) remembers having caused a great fire that killed lots of people (although fortunately it turns out it wasn't her).
Jean Greyerl's backstory has been one big Trauma Conga Line including the death of her goat and almost drowning herself.
Woolseyism: During one puzzle, the French version reminds the player the decimal mark is a comma in French, even though it's a dot in the given example.