A manufacturing method of a display substrate, a display substrate and a display device are disclosed. In the manufacturing method of the display substrate, the display substrate includes a pixel region (2670), the pixel region (2670) includes a plurality of pixel units (210) arranged in an array, and each of the pixel units (210) includes an active display region (211) and a peripheral region (212) located around the active display region (211). The manufacturing method includes: forming a plurality of spacers (220) in the peripheral region (212) through an ink-jet printing process, each of the plurality of spacers (220) includes an adhesive material (221) and at least one kind of nanoparticles (222) doped in the adhesive material (221). The manufacturing method of the display substrate can achieve applying the spacers (220) at a designated position by utilizing a precise positioning function of the ink-jet printing process, which can not only improve a utilization rate of the material for preparing the spacers (220), but also simplify the manufacturing processes. In addition, the doping of the nanoparticles (222) in the spacers (220) can achieve the functions of extending the life of a display device such as moisture absorption, heat dissipation, and gas absorption.