The invention discloses a preparation method of casein hydrolysis peptide. The preparation method comprises the following steps: (1) dissolving casein with water to prepare a casein solution of which the mass concentration is 5-15wt%, and adjusting the pH value to 5-10: (2) adding an enzyme and an enzyme activity keeping agent into the casein solution to perform hydrolysis at 45-75 DEG C for 1-5 hours; (3) performing enzyme inactivation for 5-15 minutes in a water bath of 85-100 DEG C to obtain an enzyme hydrolysis solution; (4) adjusting the pH value of the enzyme hydrolysis solution to a 4-5 isoelectric point for deposition, and performing centrifugal separation; and (5) performing ultrafiltration and freeze-drying. According to the preparation method of the casein hydrolysis peptide, disclosed by the invention, the casein hydrolysis peptide is moderate in hydrolysis degree, high in content of oligopeptide, low in content of free amino acid, low in allergenicity, and easy to digest and absorb. The raw materials used in the preparation method contain no harmful substances, cannot pollute environment, and are wide in source, the preparation process is simple, and the product has the effects of improving immunity, maintaining beauty and keeping young, promoting cell growth, and resisting aging.