Provided are an organic light-emitting display substrate and a manufacturing method thereof, and an organic light-emitting display device. The display substrate includes a substrate and an organic layer, an anode layer, an organic functional layer, and a cathode layer arranged sequentially on one side of the substrate. In a passive matrix organic light-emitting display area, the organic layer comprises a plurality of grooves spaced apart along a first direction and extending along a second direction, the anode layer comprises a plurality of first anodes arranged in an array and a plurality of shielding portions. Each shielding portion partially overlaps with an orthographic projection of a groove on the substrate to form a partition groove. The cathode layer comprises a plurality of cathode strips and cathode material portions. Each cathode material portion is located within a partition groove and is not connected to an adjacent cathode strip.