The invention provides a flexible display substrate and manufacturing method thereof. The manufacturing method for flexible display substrate patternizes the inorganic layer between the two organic layers on the flexible substrate so that the two organic layer contacting each other at the gaps on the inorganic separation layer, so as to avoid the potential damage on the films during the subsequent laser-lift-off process. The method is simple and the manufacturing process is efficient. The flexible display substrate of the present invention uses a structure of stacked four thin film layers to realize effective water/oxygen blocking, thus avoiding display devices' eroding. The inner two organic layers of the flexible substrate contact each other directly at the gaps of the inorganic separation layer, and the high adhesion strength between the upper and lower film layers of the flexible substrate helps avoiding film-peeling inner the stacked structure.