The invention provides an antifreeze polypeptide and preparation method thereof. The method takes cow leather collagen as raw material and obtains the specific antifreeze polypeptide by screening and optimizing the enzymolysis condition of alkali protease, separating and purifying. The antifreeze polypeptide has the molecular weight of 2107Da and the amino acid full sequence of Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg. The invention breaks through the research thinking and method of antifreeze protein at home and abroad, overcomes the limitation of the number of purified antifreeze protein in natural organism and the safety worry of international FDA organization for transgene antifreeze protein in the food application, obtains the food-source high-efficiency antifreeze polypeptide, and lays the theoretical foundation for developing the antifreeze polypeptide based on the food source and exploring the wide application of the antifreeze polypeptide in the aspects of food and medical science.