A preparation method of a color film layer, a display substrate and a preparation method thereof. The display substrate includes a bearing layer and repeating units, each repeating unit including a green film layer, a red film layer and a blue film layer, which are provided on the bearing layer and directly contacting a first surface of the bearing layer. On a plane parallel to the first surface of the bearing layer, in each repeating unit, the red film layer is provided on a first side of the green film layer, the blue film layer is provided on a second side of the green film layer opposite to the first side; one of the red film layer and the blue color film layer covers part of the green film layer; one part of the green film layer does not overlap with the red film layer and the blue film layer.