A display substrate and a manufacturing method and a display device are provided. The display substrate includes: a base substrate, a first insulation layer, a first electrode pattern, a connecting electrode pattern, a second electrode, a light-emitting functional layer, and a first filling layer. The second electrode is connected with the connecting electrode pattern, the second electrode and the first electrode pattern are spaced apart from each other. The light-emitting functional layer is located between the first electrode pattern and the second electrode. The first filling layer is located between the connecting electrode pattern and the first electrode pattern. The first filling layer and the light-emitting functional layer are different layers; a portion of the first insulation layer that is located between the first electrode pattern and the connecting electrode pattern has a groove, and the first filling layer is at least partially located in the groove.