The present invention discloses a display substrate and a manufacturing method thereof and a display device. The display substrate comprises a base substrate and color matrix patterns and photo spacers provided on the base substrate, edges of the adjacent color matrix patterns are overlapped, and the photo spacers are provided above the overlapped parts of the adjacent color matrix patterns. By adopting such a structure, the thickness of the photo spacers can be reduced, the purpose of increasing the cell gap of the display device is achieved while providing thinner photo spacers, and the cell gap of the display device is increased.