The present disclosure discloses a pixel defining layer, a display substrate, and manufacturing methods thereof, and relates to the field of display technology. The pixel defining layer includes: a defining layer body, and a temperature-sensitive polymer layer located on an inner side surface of the defining layer body. The temperature-sensitive polymer layer has a solution temperature threshold, and the hydrophilicity and hydrophobicity of the temperature-sensitive polymer layer are different under temperatures of the temperature-sensitive polymer layer higher and lower than the solution temperature threshold. By changing the temperature of the temperature-sensitive polymer layer to be higher or lower than the solution temperature threshold thereof, the temperature-sensitive polymer layer is converted from hydrophilic to hydrophobic. Thus, the pinning effect of the raw material solution on the inner side surface of the defining layer body is reduced and the thickness uniformity of the pixel layer is improved.