The invention belongs to the technical field of experiments and relates to a preparation method of a cross section of hairtail sagitta. The preparation method comprises the following steps: 1), marking a center position of the sagitta, 2) clamping the sagitta on a pair of fine-end self-locking forceps, 3) embedding the sagitta with a hot melt adhesive, 4) grinding the sagitta to a marking line, 5) tightly attaching and bonding a grinding surface of the ground sagitta to a glass slide, and 6) grinding from the front end to the center of the sagitta, stopping grinding when an annual ring appears, and allowing a grinding plane to be perpendicular to a cephalocaudal axis of the sagitta all the time. According to the method, the sagitta is embedded with the hot melt adhesive; the hot melt adhesive exerts effects of resin embedding, fixing and protection and can be cooled to the extent that the grinding can be performed in several minutes; a resin embedding material is saved; moreover, the working efficiency is improved.