Provided are a display motherboard, a preparation method thereof, a display substrate and a display device. The display motherboard includes a plurality of display substrate regions and a cutting region located at a periphery of each of the plurality of display substrate regions; the display motherboard includes a driving structure layer arranged in each of the plurality of display substrate regions and a marking structure layer arranged in each cutting region, wherein the marking structure layer includes a cutting mark layer; and a planarization layer arranged on the driving structure layer and the marking structure layer, and covering the marking structure layer.