Provided are an organic light-emitting display substrate and a manufacturing method thereof, and an organic light-emitting display device. A display area of the display substrate includes at least one opening. The display substrate includes a substrate, and an organic layer, a first inorganic layer, an anode layer and an organic functional layer that are sequentially arranged on one side of the substrate. The inorganic layer and the organic layer has at least one annular partition groove corresponding to each opening. A width of an orthographic projection of the notch of the annular partition groove on the substrate is smaller than that of an orthographic projection of the annular partition groove on the substrate. The functional layer includes a first organic functional material portion located outside the annular partition groove and a second organic functional material portion located inside the annular partition groove that are not connected.