A display substrate, a manufacturing method thereof, and a display device are provided. According to embodiments of the present disclosure, the manufacturing method of a display substrate comprises: fabricating a gate electrode, a gate electrode insulating layer, and a semiconductor active layer sequentially on a base substrate; fabricating a first etching stopping layer and a second etching stopping layer on the base substrate with the semiconductor active layer fabricated thereon, wherein the first etching stopping layer is disposed in a display area of the display substrate, the second etching stopping layer is disposed in a peripheral area of the display substrate, and the second etching stopping layer is a non-transparent layer; and fabricating source/drain electrodes by a patterning process, on the base substrate with the first and second etching stopping layers fabricated thereon, wherein the second etching stopping layer is used as an alignment marker in fabricating the source/drain electrodes.