The invention discloses a cryoprotectant and a preparation method thereof, and application of the cryoprotectant in coccidiosis oocysts. The cryoprotectant comprises the following components: lauric acid, hydrogenated phosphatidylcholine, sodium fluoride, ammonium chloride, asparaginic acid, proline, cane sugar, sodium dihydrogen phosphate, disodium hydrogen phosphate, a PBS buffer solution and water for injection. The cryoprotectant is reasonable in formula, ice crystal recrystallization in the cryopreservation process can be effectively inhibited, ice crystal morphology is regulated and controlled, the ice crystal growth rate is reduced, and damage of ice crystals to coccidiosis oocysts is reduced; and meanwhile, phospholipid molecules can be adsorbed to the walls of the coccidiosis oocysts, so the ice damage resistance of the coccidiosis oocysts is improved, and the coccidiosis oocysts can be stored in liquid nitrogen for a long time. Compared with a traditional cryoprotectant, thecryoprotectant of the invention has the advantages that the cryoprotectant does not contain toxic molecules such as DMSO and components such as expensive serum, avoids the toxic damage of DMSO to coccidiosis oocysts, and is reduced in production cost, effectively improved in applicability in the field of coccidiosis vaccines and beneficial to wide popularization and application.