A display substrate, a method for preparing the same and a display device are provided. The method for preparing the display substrate includes: providing a base substrate, in which the base substrate includes a first region and a second region; forming a first structure in the first region using a first mask and a first photoresist; forming a functional material layer at a side, which is away from the base substrate, of the first structure; and patterning the functional material layer using a second mask and the first mask to form a functional layer in the second region.