A display substrate including a base substrate and a pixel defining layer is provided. The pixel defining layer includes a first defining layer and a second defining layer, which define a plurality of lower openings and a plurality of upper openings corresponding to the plurality of lower openings, respectively. An orthographic projection of a bottom surface of each upper opening on the base substrate covers that of a corresponding lower opening on the base substrate, orthographic projections of bottom surfaces of the plurality of upper openings on the base substrate have an equal area. The plurality of lower openings include a first lower opening, a second lower opening and a third lower opening, which form a first cavity, a second cavity and a third cavity respectively together with a corresponding upper opening.