A display substrate, a manufacturing method thereof and a display device are provided. The manufacturing method of the display substrate includes: forming a plurality of display substrate regions and a plurality of connection lines on a mother substrate, wherein each of the display substrate regions includes a plurality of peripheral wirings, and the plurality of peripheral wirings of each of the display substrate regions are electrically connected with each other through at least one of the plurality of connection lines; and cutting the mother substrate according to the plurality of display substrate regions to form a plurality of display substrates, wherein the plurality of peripheral wirings of each of the cut display substrates are disconnected from each other.