Embodiments of the invention provide a substrate, a preparation method of the substrate and a display device, relate to the technical field of display, and can solve the problem that the thickness ofa light-emitting functional layer formed in an opening region of an existing pixel definition layer is not uniform. The preparation method of the substrate comprises the following steps of forming a pixel definition layer thin film on a substrate, wherein the material of the pixel definition layer thin film comprises a photoresist, and a hydrophilic material, elastic materials and auxiliary particles doped in the photoresist; the auxiliary particles are used for releasing gas during decomposition, and the elastic material is used for expanding the pixel definition layer thin film when the auxiliary particles release the gas; the molecular weight of the hydrophilic material, the photoresist and the elastic material is larger than that of the auxiliary particles; heating the pixel definitionlayer thin film, and enabling the auxiliary particles to move in the direction far away from the substrate; and processing the pixel definition layer thin film to form a pixel definition layer, and enabling the auxiliary particles to be decomposed to release gas. The preparation method is used for preparing the pixel definition layer.