A display substrate, an ink jet printing method and a display apparatus are provided. The display substrate includes a base substrate and a pixel define layer disposed on the base substrate, wherein the pixel define layer includes first define layers and a second define layer, a printing region is formed on the base substrate between the first define layers, and the second define layer is disposed on the printing region and divides the printing region into at least two sub-printing regions, and a height of the first define layers is greater than that of the second define layer in a direction perpendicular to the base substrate.