The present disclosure provides a display substrate and a manufacturing method thereof, and a display device, belongs to the field of display technology. The method includes forming a first thin film transistor, which includes: forming a first gate of the first thin film transistor on a base substrate through a patterning process; forming a first gate insulating layer on a side of the first gate distal to the base substrate; sequentially forming a first semiconductor material layer, a second gate insulating layer and a second gate metal layer on a side of the first gate insulating layer distal to the base substrate, and forming a pattern including an active layer of the first thin film transistor, a pattern of the second gate insulating layer and a second gate of the first thin film transistor through a patterning process.