The invention discloses an organic light-emitting superlattice thin film, a preparation method and application thereof, and belongs to the field of organic semiconductor photoelectric materials. The organic light-emitting superlattice film is an organic light-emitting film formed by alternate epitaxial growth of two kinds of two-dimensional organic molecules on the substrate surface, and the two-dimensional organic molecules are selected from perylene-3,4,9,10-tetracarboxylic dianhydride, N,N'-Dimethyl-3,4,9,10-perylenetetracarboximide, N,N'-dioctyl-3,4,9,10-perylenedicarboximide and 3 ,4,9,10‑Tetracarboximide. The preparation method is as follows: the growth source material of the first two-dimensional organic molecule and the substrate are placed in different positions of the tube furnace, and the first layer of organic light-emitting film is epitaxially grown on the surface of the substrate; the growth source material is replaced by the second Two-dimensional organic molecules, grow the second layer of organic light-emitting thin film; repeat the replacement of growth source materials, and alternately grow multi-layer organic light-emitting thin films, that is, organic light-emitting superlattice thin films. The organic light-emitting superlattice thin film has high quality and high luminous intensity, and can be used as a light-emitting layer of an organic light-emitting field effect transistor.