A display substrate and a manufacturing method thereof, and a display device are provided. The manufacturing method of the display substrate includes: forming an insulation layer on a base substrate, the base substrate including a display area and a peripheral area; and forming a planarization film on the insulation layer; performing a patterning process to the planarization film to form a planarization layer with a first thickness in the display area, a planarization layer with a second thickness in the peripheral area, and a first via hole in the planarization layer with the second thickness, the second thickness being less than the first thickness, and performing an etching process on the peripheral area to thin or remove the planarization layer is with the second thickness, and forming a second via hole corresponding to the first via hole in the insulation layer.