Abstract: Chicken lung is generally utilized as a low-value feed in poultry slaughtering industry. In order to increase the added value of chicken lung, this study determined the proximate chemical composition of chicken lung and prepared antioxidant peptide by enzymatic hydrolysis. Firstly, using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging rate and crude peptide content as indexes, the effects of the kinds of enzyme, pH, enzyme supplemental levels and hydrolysis time on antioxidant peptide activity were investigated by single factor experiment. Secondly, based on the results, Box-Behnken response surface designed was used to optimize the reaction conditions for enzymatic hydrolysis of chicken lung antioxidant peptide. The main influencing factors were pH, enzyme supplemental levels and hydrolysis time (P<0.05). The optimal conditions were obtained as follows: pH3.60, enzyme supplemental levels 4200 U/g, hydrolysis time 5 h. The DPPH· scavenging rate and crude peptide content in the validation test were 94.96% and 88.42%, respectively. Thirdly, compared with the supernatant of chicken lung homogenate, the free amino acid content of the crude peptide increased significantly (P<0.05). Finally, among the ultrafiltration products, the <3 kDa component had the strongest antioxidant capacity while the proportion of molecular weight <1000 Da was 86.70%. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) sequencing revealed that peptides with water residue ratio ≥50% were 60.97%. Meanwhile, Val and Leu accounted for 48.78% of the total N-terminal frequency of occurrence from peptide. The present results could provide theoretical and technical support for the comprehensive utilization of chicken lung.