Embodiments of the present invention disclose a method of manufacturing a flexible display substrate. The method includes steps of: forming, on a support substrate, a separation pattern layer comprising a plurality of separation units arranged at intervals; forming an adhesion layer on the support substrate formed with the separation pattern layer; forming a flexible base substrate on the adhesion layer and forming a display element on a side of the flexible base substrate opposite to the adhesion layer; performing a peeling preprocessing on the support substrate, and peeling the flexible base substrate from the support substrate to form the flexible display substrate.