The present disclosure relates to a display substrate, a method for preparing the same, and a display device. The display substrate of the present disclosure includes a base substrate, a pixel definition layer located on the base substrate, and a first pixel and a second pixel that are adjacent to each other and defined by the pixel definition layer, in which a spacer function layer for blocking hole transport between adjacent pixels is arranged at at least a part of a contact interface between the second hole transport layer in the second pixel and the light function layer in the first pixel. By providing the spacer function layer, the present disclosure effectively prevents the migration of holes between the hole transport layers of adjacent pixels or between the hole transport layer and the light emitting layer, thereby avoiding accompanying light emission between adjacent pixels.