A display substrate, a manufacturing method and a display device are provided. The display substrate includes: a base substrate and a plurality of sub-pixels; a pixel definition layer arranged on the base substrate and including a plurality of pixel openings, a region where the pixel opening is located being an active light-emitting region of a corresponding sub-pixel; a plurality of post spacers arranged at a side of the pixel definition layer away from the base substrate and configured to support a mask in an evaporation process of the display substrate; and a reflection layer arranged between the post spacer and the base substrate. An orthogonal projection of each post spacer onto the base substrate at least partially overlaps an orthogonal projection of the reflection layer onto the base substrate.