The present disclosure provides a display substrate, a method for preparing the same, and a display device. The display substrate includes an insulating pattern whose surface layer is composed of a hydrophobic fluorine-containing material, so the organic ink used to prepare a light emitting layer does not overflow outside a pixel area. The method for preparing the display substrate includes: forming a rheological insulating material layer on the base substrate; curing the rheological insulating material layer, and patterning the cured insulating material layer to obtain an insulating pattern; heating the insulating pattern, to gather the hydrophobic insulating structure on a surface of the insulating pattern away from the base substrate; heating the gathered hydrophobic insulating structure to melt it, and then cooling the molten hydrophobic insulating structure to form a metal pattern on the surface of the insulating pattern.