Provided are a display substrate and a manufacturing method thereof, and a display device. The display substrate includes: a base substrate; a drive circuit layer formed on the base substrate and including an interlayer dielectric layer at the display region and the transition region; display parts at the display region, including a first electrode and a pixel definition portion sequentially formed on the interlayer dielectric layer; a first packaging dam and a second packaging dam at the transition region and around the opened region, each including a protective portion and a blocking portion sequentially laminated on the interlayer dielectric layer. The second packaging dam is at a side of the first packaging dam away from the display region, and a thickness of the second packaging dam is greater than that of the first packaging dam.