Provided are a display substrate and a preparation method thereof, and a display apparatus. The display substrate includes a plurality of display units; the display unit includes a driving structure layer, a light emitting structure layer and a color filter layer which are located in the display area and sequentially arranged on the substrate; the light emitting structure layer comprises a plurality of light emitting structures each including a pixel define layer and an organic light emitting layer; the organic light emitting layer is located in an opening area of the pixel define layer and on the pixel define layer; the color filter layer includes a plurality of light filters; the display substrate includes a spacing area located in the display area, the spacing area being located between opening areas of pixel define layers of adjacent light emitting structures.