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!
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.