A display substrate and a manufacturing method thereof, and a display device are disclosed. The display substrate includes a base substrate, lead wires, a plurality of contact pads, a first insulation laminated layer, and a second insulation laminated layer. The base substrate includes a display region, a dam region at least partially surrounding the display region, a transition region on a side of the dam region away from the display region, and a first bonding region and a bonding peripheral region on a side of the transition region away from the dam region. The plurality of contact pads is in the first bonding region and configured to be electrically connected to the lead wires. The first insulation laminated layer is in the first bonding region. The second insulation laminated layer is in the bonding peripheral region.