Abstract: A display substrate and a manufacturing method thereof, and a display device are provided. The display substrate includes: a base substrate, a plurality of sub-pixels, a pixel defining layer, and a planarization layer. The pixel defining layer includes a main body portion, the main body portion includes a first main body sub-portion and a second main body sub-portion, the second main body sub-portion protrudes relative to the first main body sub-portion to form a separation structure, at least one of the plurality of film layers is broken at the separation structure, an orthographic projection of a part of the light-emitting functional layer in the opening on the base substrate at least partially overlaps with an orthographic projection of a part of the light-emitting functional layer located on a side of the main body portion away from the base substrate on the base substrate.