A display substrate includes a base substrate, a thin film transistor array layer, a planarization layer, a first electrode and a pixel definition layer, the pixel definition layer defining a plurality of pixel openings, each pixel opening includes a first edge and a second edge adjacent to each other, the pixel definition layer includes a first pixel definition layer parallel to the first edge and a second pixel definition layer parallel to the second edge. A surface of the first pixel definition layer away from the base substrate is located at a level lower than a surface of the second pixel definition layer away from the base substrate, a groove parallel to the first edge is arranged in a surface of the planarization layer away from the base substrate, at least a part of the first pixel definition layer is arranged in the groove.