The invention provides a display substrate and a manufacturing method thereof, and a display device. The display substrate comprises: a base, and gate lines and data lines, the data lines and the gate lines intersect with each other and are insulated from each other, the gate lines are electrically connected to a gate driver, the data lines are electrically connected to a source driver, the gate driver is provided at one side opposite to the source driver; gate signal lead-in lines, which are arranged parallel to the data lines; and an interlayer insulation layer provided between a layer in which the gate signal lead-in lines are arranged and a layer in which the gate lines are arranged, one end of each gate signal lead-in line is electrically connected to the gate driver, the other end thereof is connected to a gate line through a via penetrating the interlayer insulation layer.