Crowning Moment of Funny: Luke (for reasons unexplained) decided to keep the Flora mask that Don Paolo was wearing. The end credits show him wearing it on the train. (Actually, he's seen messing around with Don Paolo's masks during the credits of every game.)
Made even funnier by Flora's reaction.
Maybe he kept it as a souvenir of this adventure?
Also, the sort of Easter eggs you can find by tapping. Tap Babette on the train when she is seated in the restaurant car, and if, after solving 100, you tap the bridge. Luke says, "The Herzens should have invested a little of that legendary wealth into repairing this ratty bridge."
Any of Luke and Layton's reactions to a bad tea mix.
Luke: P-Professor! There's smoke coming off this tea!
Other conversations detail congealing tea (with Luke commenting that they might have to eat it like a pudding), foaming tea (which Layton comments is not fit for human consumption and can perhaps be used to instead clean floors), tea that looks like mud, tea that tastes of old shoe, murky tea, tea that's rank, tea that is so bad it fails words, tea that smells funny and that neither of them are brave enough to drink... It's amusing because it's about the only time you ever hear Layton be flat-out critical of anything (then again it can be inferred he's the one who made the teas he's being so harsh about) or less than a gentleman by trying to get Luke to drink the tea first. One of them (using Joy Luck Clover, Belle Tart Seeds, and Brisk Berry) probably takes the cake of unique and entertaining reactions because it literally sucks the joy out of the two of them:
Layton: Oh, this won't do at all.
Luke: Ugh, this tea is making me sad. One sip was enough to make me want to cry.
Layton: I don't think I've ever come out of teatime wearing anything but a smile... until now.
Luke: Believe me, Professor, you're not the only one frowning.
Luke's reaction to meeting Chelmey for the first time — and thinking he's Don Paulo! Cue Layton having to apologise on Luke's behalf while the scamp tugs at Chelmey's face, trying to tug it off because he thinks it's a mask!
During the credits, Layton's reaction on seeing Luke interacting with the cabaret woman.
Or when they're opening the box. Luke starts screaming in an exaggerated way, and the fact that the situation is supposed to be tense makes it even more hilarious.