The invention discloses a display substrate, a manufacturing method thereof, and a display device, and relates to the field of display technology, and aims to solve the problem that the picture display quality of the display device is lowered due to uneven cell thickness of the display substrate. The display substrate includes a first substrate and a second substrate oppositely arranged, the first substrate includes a first area and a second area, and the total thickness of the functional layers located in the first area on the first substrate is greater than that located in the second area. The total thickness of the functional layer is thin; a thickness compensation layer is provided between the first substrate and the second substrate, the thickness compensation layer corresponds to the first area, and the total thickness of the thickness compensation layer and the functional layer in the first area is the same as that of the second area The total thickness of the inner functional layer is equal. In the present invention, a thickness compensation layer corresponding to the first region is arranged between the first substrate and the second substrate, so that the cell thickness of each region of the display substrate is uniform, so as to improve the image display quality of the display device.