A display substrate includes a base substrate, a pixel definition layer, an isolation layer, and an isolated layer. The pixel definition layer includes pixel openings penetrating through the pixel definition layer. The isolation layer is not overlapping with the pixel openings. The isolation layer includes a first isolation portion and a second isolation portion. A slope angle formed by at least one side edge and a bottom edge in a cross section of the second isolation portion is an obtuse angle. There is a gap between the first isolation portion and the second isolation portion at the obtuse angle. The isolated layer includes a first isolated portion covering the isolation layer, and a second isolated portion and a third isolated portion on both sides of the first isolation portion respectively. The thickness of the first isolation portion is larger than the thickness of the isolated layer.