A display substrate, a manufacturing method thereof, and a display device are provided. The display substrate includes: a base substrate; an anode conductive layer; a cathode conductive layer, wherein the cathode conductive layer and the anode conductive layer are at a same side in a thickness direction of the base substrate; and an organic functional layer between the anode conductive layer and the cathode conductive layer, wherein the display substrate comprises first sub-pixel units and second sub-pixel units that are different in color category, the organic functional layer in the first sub-pixel units comprises a first light-emitting layer, the organic functional layer in the second sub-pixel units comprises the first light-emitting layer, a first electron blocking layer and a second light-emitting layer that are sequentially laminated in a direction away from the anode conductive layer, and a light-emitting color of the first light-emitting layer is different from a light-emitting color of the second light-emitting layer.