A display substrate, a manufacturing method thereof, and a display device are provided, and the manufacturing method includes: providing a base substrate; forming a pixel definition layer on the base substrate; oxidizing the pixel definition layer, in which a surface of the pixel definition layer distal to the base substrate is partially oxidized, such that the pixel definition layer includes a main layer proximal to the base substrate and an oxide layer distal to the base substrate; curing and molding the pixel definition layer, and patterning the pixel definition layer to form a pixel definition layer pattern.