The invention belongs to the technical field of dairy product processing, and particularly discloses a preparation method for high-activity peptide acidified milk. The method comprises the steps: S1, preparation of a leavening agent; S2, inoculation; S3, two-stage fermentation. According to the method, fermentation strains are screened and are compounded according to a certain ratio, and a two-stage fermentation method is adopted, so that the coordinate symbiosis among various strains gives full play to own characteristic advantages, the acid production, aroma producing and proteolysis capabilities of the strains tend to an optimal state, acidified milk obtained by production has more small molecule peptide than traditional acidophilus milk. Casein and whey protein are both subjected to advanced degradation during a fermentation process, so as to be easily digested and absorbed by a human body, the prepared high-activity peptide acidified milk has the healthcare functions of lowering serum cholesterol, reducing blood glucose, resisting hypertension, resisting oxidation, boosting immunity, improving intestinal function, and the like.