Embodiments of the present invention provide a substrate and a fabrication method thereof, and a display device. The substrate includes a base and a pixel defining layer in which a plurality of openings are formed, the pixel defining layer includes a first defining layer, a second defining layer and a third defining layer sequentially stacked on the base, wherein, the second defining layer is configured as a conductive layer, and an orthogonal projection of the second defining layer on the base is at least partially located outside an orthogonal projection of a side of the third defining layer close to the base on the base.