The present invention discloses a pixel defining layer, a preparation method thereof and a display panel. The invention aims to improve the film formation uniformity of an OLED (Organic Light EmittingDiode) light emitting layer, improve the light emitting effect of the OLED light emitting layer, and prolong the service life of a display device. The pixel defining layer provided by one embodimentof the present invention is disposed on a substrate; the pixel defining layer comprises at least four pixel defining sub-layers which are composed of hydrophilic pixel defining layers and hydrophobicpixel defining layers which are alternately stacked on the substrate; the pixel defining sub-layer adjacent to the substrate is a hydrophilic pixel defining layer; and in a direction perpendicular tothe substrate, the pixel defining sub-layer farthest from the substrate is a hydrophobic pixel defining layer.