The invention relates separation preparation and application of an Arcainflata Reeve oxidation-resistant active peptide. The separation preparation method comprises the following steps: peeling and cleaning Arcainflata Reeve, carrying out tissue homogenization, carrying out hydrolysis technique screening and optimization, centrifugating, taking the supernate, and carrying out freeze-drying to obtain an active polypeptide component; and sequentially carrying out ultrafiltration interception concentration, ion-exchange chromatography, gel filtration and high-efficiency exclusion chromatography on the active component to finally obtain the oxidation-resistant active polypeptide with the purity of 99%. Multiple modern protein purification techniques are creatively utilized to separate the active polypeptide with definite material basis from Arcainflata Reeve hydrolysate; the molecular weight of the active polypeptide is 17578.31Da; and the active polypeptide contains the following secondary structures: 40.8% of alpha-spiral, 24.7% of beta-fold, 15.9% of beta-corner and 18.6% of random coil. The polypeptide has obvious oxidation-resistant activity; the in-vitro removal DPPH, ABTS and hydroxy free radical IC50 values are respectively 3.89, 0.22 and 10.77 mg/ml; and the polypeptide can prolong the average life of the Caenorhabditis elegans by 18.29%. Therefore, the active peptide can be used for development and application of antioxidants, medicinal cosmetics or health products.