A display substrate, a manufacturing method thereof and a display device are provided. The display substrate includes a base substrate, and a shift register unit, a first clock signal line and a second clock signal line which are on the peripheral region of the base substrate; the first clock signal line and the second clock signal line extend along a first direction; an active layer of the first control transistor, an active layer of the second control transistor, and an active layer of the third control transistor respectively extend along a second direction, and the active layer of the first control transistor, the active layer of the second control transistor, and the active layer of the third control transistor are on a side of the first clock signal line and the second clock signal line close to the display region, and are arranged side by side in the first direction.