A display substrate, a manufacturing method thereof, and a display device are provided. The display substrate includes: a base substrate at least including a pixel area and a hole area; a plurality of sub-pixels arranged on the base substrate and located in the pixel area; a hole in the hole area; a first barrier dam arranged between the sub-pixels and the hole and at least partially surrounding the hole; an organic material layer including at least one film layer, wherein an orthographic projection of the organic material layer on the base substrate falls within the pixel area; and a filling structure, wherein at least a portion of the filling structure is arranged between the hole and the first barrier dam, and the filling structure and the at least one film layer of the organic material layer are located in the same layer and include the same material.