A display substrate and preparation method thereof, an ink-jet printing method and a display device are provided. The display substrate includes a display area, a peripheral area, and a pixel definition layer; the pixel definition layer including: a display pixel definition region and an assistant pixel definition region surrounding the display pixel definition region; the display pixel definition region includes a plurality of first pixel definition units; the assistant pixel definition region includes a plurality of second pixel definition units, and opening areas of the second pixel definition units are respectively greater than those of the first pixel definition units; the display pixel definition region is in the display area, the assistant pixel definition region is in the peripheral area, the plurality of first pixel definition units is configured to form display pixels, and the plurality of second pixel definition units is configured to form assistant pixels.