A method of manufacturing a display substrate, a display substrate and a display device are provided. The manufacturing method includes: forming an organic material layer in a display area (10) and a non-display area (11); forming an auxiliary layer (24, 25) in the non-display area (11) before forming the organic material layer. The performances of the auxiliary layer (24, 25) are changed after heating, to allow a portion of the organic material layer corresponding to the auxiliary layer (24, 25) can be easily stripped off. The method further includes removing the portions of the organic material layer corresponding to the auxiliary layer by a heating process.