A method for preparing a pixel defining layer (PDL), an organic light-emitting diode (OLED) substrate prepared by the PDL, and a display device are provided, which can overcome the problem that the conventional PDL comprising hydrophilic and hydrophobic materials involves a complicated process. The method for preparing the PDL comprises: step 1: forming a mixed solution on a substrate to form a mixed solution film, in which the mixed solution includes a hydrophilic material, a hydrophobic material and a solvent; step 2: performing heat treatment on the mixed solution film to form a pixel defining material layer in which the content of the hydrophilic material on the upper portion is less than that of the hydrophilic material on the lower portion and the content of the hydrophobic material on the upper portion is more than that of the hydrophobic material on the lower portion; and step 3: forming a pattern of the PDL on the pixel defining material layer by a patterning process. This method for preparing a PDL is simple and easy to realize.