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 a first electrode layer between the base substrate and the pixel defining layer. The pixel defining layer defines a plurality of sub-pixels on the base substrate, the first electrode layer comprises a plurality of first electrodes, and the plurality of first electrodes being separated from each other by gaps, and an orthographic projection of the pixel defining layer on the base substrate covers an orthographic projection of the gap on the base substrate.