A display substrate, a manufacturing method thereof and a display device are provided. The display substrate includes a base substrate, a pixel defining layer and at least one photo spacer. The pixel defining layer is disposed on the base substrate and includes a plurality of openings, the at least one photo spacer is disposed on a side of the pixel defining layer away from the base substrate. A distance from any point at a bottom of the at least one photo spacer contacting the pixel defining layer to an upper edge of a side wall of the plurality of openings is greater than or equal to 5 μm.