The invention discloses a preparation method of a frozen diluent of sheep semen and an application of the preparation method. The preparation method of the frozen diluent of the sheep semen comprisesthe following steps: firstly, accurately weighing tris(hydroxymethyl) aminoethane, dihydrate trisodium citrate, fructose, an inositol compound, ethanediol or glycerinum, and oxytocin, adding the materials into ultrapure water, dissolving, adding a fresh yolk centrifuged liquid supernatant, penicillin and streptomycin, mixing evenly, fixing the volume after adjusting the pH value to 6.8-7.2, thus obtaining the frozen diluent of the sheep semen. The inositol compound is used to replace an antifreeze protein, the defect of the application of the antifreeze protein is avoided, meanwhile, the oxytocin is added when the semen is conveyed, and the sperm vitality and the acrosomal integrity are improved, so that the fertility rate is increased.