A display substrate, a manufacturing method thereof and a display device are provided. The display substrate includes a display region, a peripheral region a plurality of signal lines configured to provide signals to the display region; a plurality of contact pads configured to be electrically connected with the plurality of signal lines; at least two conductive connection lines provided on a side of the plurality of contact pads facing away from the display region. The at least two conductive connection lines include a first conductive connection line closest to the plurality of contact pads in a first direction from the display region to the peripheral region. At least part of the plurality of contact pads are provided in a first conductive layer, the first conductive connection line is provided in a second conductive layer, and the first conductive layer and the second conductive layer are different layers.