The invention discloses an equus oocyte vitrification cryopreservation fluid, a cryopreservation method and an application. The equus oocyte vitrification cryopreservation fluid comprises a base solution (BS), an equilibrium solution (ES) and a vitrification solution (VS), wherein the VS is composed of the following components by volume percent: 10-20% of glycol (EG), 6-10% of dimethyl sulfoxide (DMSO), 6-10% of propylene glycol (PROH), 15-25% of inactivated fetal calf serum (NCS), 8-15% of carboxylate E-polylysine (COOL-PLL) and the balance of an HEPES solution. Each liter of the VS includes 0.3-0.8mol of saccharose. The invention has the beneficial effects that the vitrification cryopreservation fluid has low cytotoxicity, high permeability and obvious vitrification effect, meanwhile can obviously reduce the generation of ice crystals in freezing and re-warming processes, can protect cells from physical injury, can increase the survival rate of oocyte, is suitable for the vitrification cryopreservation of equus oocytes or embryos of horses, donkeys and the like, and is beneficial to the popularization and application of artificial insemination and embryo transplantation technology.