Provided is a display substrate, a manufacturing method thereof and a display panel. The display substrate includes a base substrate, a display region and a bonding region located at a side of the display region on the base substrate, wherein the bonding region includes a first protective layer and a bonding electrode disposed on the base substrate; the first protective layer is provided with a groove; the bonding electrode is at least partially disposed in the groove; in a direction parallel to the base substrate, the groove, the bonding electrode and the first protective layer extend to an edge of the bonding region in a direction away from the display region; and a material of the first protective layer is less hard than a material of the bonding electrode.