Puzzle #161, "Lily Pad Leapfrog 2", has 161 alternate solutions not mentioned in the game.

The Nintendo DS Start menu icons for the first four games in the series feature Layton gradually smiling more with each game before ending by pointing at something.

The bonus mode Professor Layton's London Life, featured in Professor Layton and the Last Specter, was originally going to be its own game called Fantasy Life, co-developed by Brownie Brown and Level-5. It initially began development on the Nintendo DS and had sprite art heavily reminiscent of Mother 3. After that version was cancelled, its assets were reworked as a bonus mode for Last Specter, with completely different gameplay and controls from the usual Professor Layton games and cameos from series characters.

London Life can be accessed from the main menu, much like the bonus chapter from the Japanese version of Professor Layton and the Diabolical Box. The Japanese version required the player to complete the main story to unlock it, though in the US and Australian versions, it was made available from the start. The mode was completely removed from all European versions, including the UK version, likely to avoid a delayed release from having to translate its text into multiple European languages. The button in the in-game menu to access London Life was dummied out from those versions as well.

Fantasy Life would later be expanded into its own Nintendo 3DS game by the same developers and first released in Japan in 2012.