Abstract: In this study, the eggshell membrane peptides were prepared using a two-step process involved extrution and enzymatic hydrolysis. The process parameters were determined by evaluation of the total nitrogen recovery, D-glucuronic acid yield and antioxidant activity. The optimum extrution process were 140 ℃ (temperature), 100 r/min (screw rotation speed) and 20% (moisture content). And the optimum conditions of enzymatic hydrolysis were 6 h (time), 12000 U/g (enzyme amount) and 55 ℃ (temperature). Under these conditions, the total nitrogen recovery and D-glucuronic acid yield were 60.8% and 12.4 mg/g, respectively. The ABTS, OH free radical scavenging ability and Fe²⁺ chelating ability of the shell membrane peptides were 29.46% (0.1 mg/mL), 26.58% (5 mg/mL) and 50.25% (0.4 mg/mL), respectively. And their cellular antioxidant activity reached 65% (10 μg/mL). Results on the molecular mass distribution indicated that the main components were oligopeptides (252~2435 Da), accounting for 81.8%. Analysis on the amino acid composition suggested a high level of Asp and Glu in the peptides, and the content of amino acids related with antioxidant activity was 44.70 g/100 g protein. And thus, the shell membrane peptides have great potentials as natural antioxidants in the field of food, healthy product and cosmetic.