The invention discloses a refining process of pond turtle bioactive peptides. Acetic acid immersion pretreatment is firstly adopted, expanding tissue is softened, part of impurity protein is removed, an enzymolysis method is adopted for treatment, citric acid is added to be used as an enzymolysis medium, the extraction rate of the bioactive peptides is higher than 25% while the extraction rate in the prior art is only about 10%, and defects of time consumption and energy dissipation with a conventional processing method, damage to amino acid with a general acid hydrolyzation method, high probability of racemization with an alkali hydrolysis method and the like are overcome. Besides, a multilevel membrane separation technology is combined, the selectivity is high, few by-products are produced, the adaptability is high, the purity of the separated bioactive peptides is higher than 75%, the molecular weight is smaller than 10 KD, the bioactive peptides can enter an organism circulation system completely and are easy to absorb, and however, molecular weight is not uniformly distributed and the absorption effect is poor with a commonly used centrifugal and precipitation filtering method. Desalination and debitterization are also completed in the enzymolysis, microfiltration and multilevel ultrafiltration processes, and the prepared pond turtle bioactive peptides have shallower color, don't have foreign smell, have uniform molecular weight, are safe and non-toxic, and have wide application prospect in health care of liver cirrhosis prevention, fatigue resistance, immunity enhancing and the like.