A fabrication method and a fabrication assembly for a flexible display substrate are provided. The fabrication assembly for a flexible display substrate includes: a rigid base substrate, including a first engaging structure being on a surface of the rigid base substrate; and a protective film, including a first surface and a second surface opposite to each other, and including a second engaging structure being on the first surface. The second surface of the protective film is configured to be attached to a flexible base substrate; and the rigid base substrate and the protective film are configured to be detachably connected with each other by the first engaging structure and the second engaging structure.