The present disclosure discloses a manufacturing method of a display substrate, a manufacturing method of a display panel and a display device. The manufacturing method of a display substrate comprises: performing an pre-irradiation on the display substrate with ultraviolet light after completion of alignment of an alignment layer of the display substrate, such that electrons in a metal layer of the display substrate receive energy under this irradiation of the ultraviolet light and thereby transfer, and are distributed uniformly in the alignment layer of the display area of the display substrate. A uniform distribution of charges in the whole display area would not result in the deficiency of non-uniform brightness at the edge of the display area.