The present invention discloses a flexible substrate and a manufacturing method thereof, and a display device. The method comprises: forming a first organic layer over a base substrate; forming a first buffer layer over the first organic layer; forming an inorganic layer over the first buffer layer; and forming a second organic layer over the inorganic layer. In the method of the present invention, arrangement of the first buffer layer enhances adherence between the first organic layer and the inorganic layer, thereby reducing or even eliminating risk of separation of the first organic layer and the inorganic layer in a process of bending a flexible substrate, enhancing water and oxygen resistance of the flexible substrate, and increasing quality of the substrate.