Level-5 announced the sequel in the latest Nintendo Direct conference, according to Andriasang.
Set one year after the events of the last 3DS title, Layton Kyouju to Choubunmei A no Isan will be the last game to feature the titular character, who is attempting to solve the case of a living mummy.
Professor Layton and the Last Specter, a prequel story set before the events of the original trilogy, was released on DS in 2011.
The first 3DS release Professor Layton and the Miracle Mask was a launch title for 3DS in Japan, and will be available in North America and Europe next year.
The game focuses on a wish-granting mask which has immense power and is wielded by a new villain calling himself the Masked Gentleman. It features 3D characters and environments in addition to the signature 2D art of past titles.
Developer Level-5 will also host a brand new puzzle every day for 350 days after its launch.
Watch a trailer for Professor Layton and the Miracle Mask below: