The present invention provides a novel polypeptide preservative and a preparation method thereof. The preparation method comprises the following steps: Tibet Kefir grains at the late logarithmic growth stage are inoculated into pure milk, the pure milk is subjected to sealed static fermentation at 30-40 DEG C until a part of curd is produced; the pure milk is added, a liquid fermented milk is drawn in a sterile environment and coated on a MC-solid medium and a MRS solid medium plate respectively, and the mixture is then sealed and cultured; after completion of the culture, bacterial colonies and the medium in the culture dish are added into pure milk, and at the same time, carbon and nitrogen sources are added, and the mixture is placed in a temperature of 30-35 DEG C to be fermented for 65-75 hours; and after the completion of the fermentation, the fermented milk is centrifuged to remove precipitates, the pH value of the resulted supernatant is adjusted to 2-5, the treated fermented milk is maintained at 8-10 DEG C for 60-120 min, then the fermented milk is centrifuged, the obtained precipitate is dried into a powder, and the powder is subjected to cryopreservation, thereby obtaining the natural novel polypeptide preservative. The present invention also provides a use method of the natural novel polypeptide preservative. The novel polypeptide preservative is safe and hygienic, natural and non-toxic, high-efficient, and broad in spectrum and stable in performance, and can be widely applied in the field of fresh fruit preservative.