The invention provides a casein anti-freezing agent for cryopreservation of donkey semen and a preparation method thereof. Each 100 milliliters of anti-freezing agent contains the following components: 1.0 to 3.0 grams of casein, 2.0 to 3.0 grams of glucose, 1.0 to 2.0 grams of lactose, 3.0 to 6.0 grams of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) BUFFER, 2.0 to 3.0 milliliters of glycerol, 2 to 4 milliliters of yolk, 450 to 550 thousand international units (IU) of penicillin, 4.5 to 5.5 grams of gentamicin, 20 to 30 milligrams of amphotericin B, and the balance of HANK solution. The casein anti-freezing agent solves the problems of long reproductive cycle of female animals, low artificial insemination rate, complex production operation of frozen donkey semen and high difficulty in the prior art.