The invention relates to a method for preparing a leather sample observed by a metallurgical microscope, and belongs to the technical field of leather inspection. The method comprises the following steps of: (1) adding a promoter into an unsaturated polyester resin, stirring, adding a curing agent, stirring, standing, and discharging the mixed air to obtain an immobilized resin; (2) immersing a sample into the immobilized resin, and curing to obtain a cured sample; (3) grinding both ends of the cured sample until the ends are flat, fixing in a mould, injecting the immobilized resin into the mould, curing, demoulding, and post-curing to obtain a post-cured sample block; and (4) grinding and polishing the post-cured sample block by using abrasive paper to obtain the leather sample observed by the metallurgical microscope. In the method, the unsaturated polyester resin is used as a main ingredient of the immobilized resin, and operation steps are designed according to a capillary action principle to ensure that the immobilized resin can be quickly and fully permeated into the leather sample, so the adhesion and deformation of section fiber of leather are furthest avoided.