Embodiments of the present invention provide a preparation method of a patterned film, a display substrate and a display device, avoiding falling off of a film layer occurring in the process of peeling off a photoresist layer. The preparation method of the patterned film comprises: forming a preset film layer on a surface of a preset substrate; covering the preset film layer with an isolation layer; forming a photoresist layer on a surface of the isolation layer and forming a pattern of the isolation layer with a patterning process; then removing the preset film layer which is not covered by the pattern of the isolation layer, peeling off the photoresist layer and removing the remaining, isolation layer to form a pattern of the preset film layer.