There are provided a PI substrate, a preparation method thereof and a display device. The preparation method includes: forming a first separable film at an edge region of a substrate; forming a first-layer polyamic acid (PAA) film on the first separable film and the substrate; stripping the first separable film and the first-layer PAA film that covers the first separable film to expose the edge region of the substrate; and performing imidization on the first-layer PAA film on the substrate with the edge region exposed, to enable the first-layer PAA film to be transformed into a first-layer PI film, thereby avoiding uneven film thicknesses at edges of the first-layer PI substrates.