The invention provides a preparation method of a display substrate, the display substrate and a display device. The display substrate comprises a first display region and a second display region, wherein the first display region comprises a plurality of first pixel regions, the second display area comprises a plurality of second pixel regions, the density of the first pixel regions is less than that of the second pixel regions, each first pixel region comprises a light-transmitting region and an isolation region arranged at the periphery of the light-transmitting region, the display substrateof the isolation region comprises a substrate, an organic light-emitting layer arranged at one side of the substrate and a packaging layer arranged at the side, which deviates from the substrate, of the organic light-emitting layer, the organic light-emitting layer in the isolation region is discontinuous, and the packaging layer covers the light-transmitting region. According to the technical scheme, transparent display is achieved by arranging the light-transmitting region in the first display region, the light transmittance of the first display region is improved, the optical device can bearranged in the first display region, and thus real full-screen display is achieved.