The invention relates to a sturgeon protamine and a preparation method of a sturgeon protamine polypeptide. The preparation method comprises the steps of cutting purified sturgeon sperm into a slurry;adding a mixed liquor of NaCl, NaEDTA and PMSF for homogenizing and stirring; carrying out centrifugal separation; adding sulfuric acid into a precipitate for extracting; carrying out centrifugal separation at low temperature; adjusting the pH of a filter liquor through NaOH; carrying out dialysis for desalting and ethanol precipitation; washing through acetone and a diethyl ether; vacuum freezing drying to obtain a sturgeon crude protein; purifying to obtain the sturgeon protamine; homogenizing the sturgeon protamine through a phosphate buffer solution; adding an enzyme, carrying out gamma ray irradiation, and carrying out enzymolysis to obtain an enzymatic hydrolysate; carrying out enzyme deactivation on the enzymatic hydrolysate through boiling water, cooling, adding a trichloroaceticacid solution, carrying out centrifugal separation, and vacuum freezing drying to obtain the sturgeon protamine polypeptide. The sturgeon protamine and the sturgeon protamine polypeptide provided by the invention are rich in raw materials and natural without pollution, have a high water-retaining property and an antibacterial effect, can be widely used in food, cosmetics and medicine fields, and can be specifically used as an antiheparin agent applied to an external cardiac operation.