The collision, involving a Ford Focus and a Hyundai i30, happened on the A92 between Ladybank and Freuchie shortly before 6am yesterday.
The two occupants of the Ford died at the scene. The man driving the other car was taken to Ninewells Hospital in Dundee for treatment to serious injuries.
The road was closed following the crash to allow police to carry out investigations.
The Scottish Fire and Rescue Service said they were called to the scene by the ambulance service. Crews from Glenrothes, Lochgelly, Cupar, Auchtermuchty and Dunfermline were dispatched to the scene.
Police have urged anyone with information which could help the investigation to come forward.