A display substrate, a preparation method thereof, and a display device are provided. The display substrate includes: a substrate, and a drive structure layer and a light-emitting structure layer that are sequentially stacked on the substrate and located in a display region. The display substrate further includes: M rows of scanning signal lines and M rows of light-emitting signal lines. The light-emitting structure layer includes: M rows and N columns of light-emitting structures. The drive structure layer includes: a pixel circuit array and a drive circuit array that extend in a column direction. The pixel circuit array and the drive circuit array are sequentially arranged in a row direction. The pixel circuit array includes: M rows and N columns of pixel circuits, and the pixel circuits are in one-to-one correspondence with the light-emitting structures and electrically connected to corresponding light-emitting structures.