The present disclosure provides an organic light-emitting diode display substrate and a manufacturing method thereof. The manufacturing method includes: sequentially forming a first electrode, a light-emitting layer and a second electrode on a base substrate; forming a protection layer having a first opening on a side of the second electrode distal to the base substrate; and forming a second opening in the second electrode, the second opening being located below the first opening. In the present disclosure, with the protection layer, only a portion of the second electrode below the first opening is removed, so that other portions of the second electrode are prevented from being damaged, thereby eliminating a poor display of the display device.