The invention relates to a polypeptide preparation method, and particularly relates to a preparation method of earthworm polypeptide extract, the preparation method of the earthworm polypeptide extract includes (1) pretreatment, to be more specific, taking fresh earthworms, washing clean, homogenizing, and performing ultrasonic extraction; (2) fermentation treatment, to be more specific, adding mixed strains into a solid-liquid mixture, adjusting pH, and fermenting; (3) first enzymolysis, to be more specific, adding bromelain into the fermentation mixture, adjusting pH, and inactivating enzymes to obtain an enzymolysis substance; (4) second enzymolysis, to be more specific, adding pepsin into the enzymolysis substance for enzymolysis treatment, centrifuging the enzymolysis substance for removal of slag, and collecting supernatant; and (5) ultrafiltration treatment, to be more specific, taking the supernatant collated by the step (4) for ultrafiltration with an ultrafiltration membrane, and using a nanofiltration membrane for interception of the ultrafiltered filtrate, and lyophilizing the obtained interception liquid to obtain earthworm peptides. The earthworm polypeptide extract prepared by combination of the fermentation and the enzymolysis has good color, no fishy odor and bitter taste, and has better antioxidant activity, and the method also greatly improves the extraction rate of the polypeptides.