The present disclosure provides a display substrate, a display device, and a manufacturing method of a display substrate. The display substrate of the present disclosure includes a substrate, a pixel defining layer located on the substrate, and a plurality of organic light emitting diodes arranged in an array, with a plurality of pixel accommodating holes provided in the pixel defining layer, and the organic light emitting diodes in one-to-one correspondence with the pixel accommodating holes, and located in the corresponding pixel accommodating holes; and a plurality of supporting structures in one-to-one correspondence with the pixel accommodating holes are formed on a side of the pixel defining layer distal to the substrate, and orthographic projections of the supporting structures on the pixel defining layer surround the corresponding pixel accommodating holes.