The invention relates to the field of the processing of functional foods, and particularly provides crocodilian serum protein with an antineoplastic function and a preparation method thereof. The preparation method of the crocodilian serum protein comprises the following steps of ultrasonically treating a crocodilian serum solution, adding flavorzyme into the crocodilian serum solution to carry out enzymolysis for obtaining enzymatic hydrolysate, and freeze-drying the enzymatic hydrolysate to obtain the crocodilian serum protein with the antineoplastic function. According to the crocodilian serum protein with the antineoplastic function and the preparation method thereof, the crocodilian serum protein is treated by utilizing an ultrasonic technique; the antineoplastic function of the serum protein can be improved obviously; the growth of a tumor cell is effectively controlled.