A display substrate, a manufacturing method thereof, and a display device are provided, in the field of display technology. The display substrate includes a base substrate, and a thin-film transistor, a light-emitting device, an encapsulation structure, and a conductive film layer sequentially disposed on the base substrate in a direction away from the base substrate. Since the display substrate includes a conductive film layer on a side of the encapsulation structure away from the base substrate, when the protective film layer on the side of the conductive film layer away from the base substrate is peeled off, static electricity generated by the separation of the film layer can be released to the conductive film layer, avoiding electron transition to the active layer of the thin-film transistor in the display substrate to cause offset of the threshold voltage of the thin-film transistor. The display brightness uniformity of the display substrate can be ensured.