The hydrolysate from duck egg white protein (DEWP) prepared by "SEEP-Alcalase" at degree of hydrolysis (DH) value of 21% (namely HSA(21)) exhibited high antioxidant capacity in different oxidation systems. A consecutive chromatographic method was then developed for separation and purification of HSA(21), including ion-exchange chromatography, macroporous adsorption resin (MAR) and gel filter chromatography. The final peptides "P21-3-75-B" were obtained with significantly enhanced antioxidant activity (p < 0.05). It was further confirmed that the product mainly consisted of five oligopeptides (Mr: 202.1, 294.1, 382.1, 426.3, and 514.4 Da). Furthermore, the antioxidant activity of P21-3-75-B kept stable after in vitro digestive simulation. Antioxidant capacity of the purified peptides was closely related to the molecular mass, hydrophobic amino acid residues, acidic amino acid and some antioxidant amino acids. This research provided a valuable route for producing new natural-source peptides with strong antioxidant capacity and high nutritious value for our daily intake. (C) 2014 Elsevier Inc. All rights reserved.