A display substrate includes a base substrate, a plurality of first electrodes, a first pixel defining layer, and a second pixel defining layer disposed on the base substrate, and light-emitting layers disposed in a plurality of second opening regions. The first pixel defining layer includes a plurality of first opening regions, and each of the first opening regions exposes at least a portion of a first electrode. The second pixel defining layer includes the plurality of second opening regions, each second opening regions corresponds to at least two first opening regions, and the orthogonal projections of the at least two first opening regions on the base substrate are located within the orthogonal projection of the second opening region on the base substrate. The light-emitting layers overspreads the plurality of second opening regions in a plane perpendicular to a thickness direction of the base substrate, respectively.