A display substrate, a manufacturing method thereof, and a display device are provided. The display substrate includes a base substrate and sub-pixels. The sub-pixel includes a light-emitting element which includes a light-emitting functional layer and a first electrode and a second electrode. The display substrate further includes a defining structure, a first orthographic projection of a surface, close to the base substrate, of the defining structure between adjacent sub-pixels on the base substrate is completely within a second orthographic projection of a surface, away from the base substrate, of the defining structure on the base substrate, a maximum size of the second orthographic projection is greater than that of the first orthographic projection, and the defining structure includes an inorganic nonmetallic material; the light-emitting functional layer is disconnected at an edge of the defining structure, and second electrodes of adjacent sub-pixels are at least partially continuously arranged.