A display substrate, a manufacturing method thereof, and a display device are disclosed. The display substrate includes a display region and a peripheral region encircling the display region. A sealant is disposed in the peripheral region and includes at least one corner. The display substrate further includes at least one retaining wall disposed at the at least one corner of the sealant; the at least one retaining wall is disposed in one-to-one correspondence with the at least one corner of the sealant and is located on one side of the sealant facing the display region. The retaining wall has an integral structure.