The present disclosure provides a display substrate, a fabrication method thereof and a display panel. The display substrate includes a substrate; and a pixel defining layer on the substrate. The display substrate further comprises a display area, a non-display area and a light-transmitting area, the display area and the non-display area at least partially surround the light-transmitting area, the pixel defining layer extends from the display area to the non-display area, the pixel defining layer is provided with a first opening, the first opening comprises a plurality of first sub-openings and a plurality of second sub-openings, the plurality of first sub-openings are in the display area, the plurality of second sub-openings are in the non-display area, and the plurality of second sub-openings are closer to the light-transmitting area than the plurality of first sub-openings.