Abstract: In this study, the enzymatic method was used to prepare Se-containing peptides (SeCPPs) with antioxidant activity from Cardamine enshiensis. The extraction conditions of protein of C. enshiensis (SeCP) were optimized by single-factor experiments and orthogonal test with protein extraction rate as the index. The enzymatic parameters of SeCP were optimized with DPPH∙ and ABTS⁺∙ scavenging rate as the investigated index, and ultrafiltration equipment was used to determine the best protocol for the SeCPPs. The reducing power and Fe²⁺ chelating ability of Se-containing peptides were investigated. The results showed that the optimal conditions for the extraction of SeCP were: 0.1 mol/L NaOH solution, liquid to material ratio 45:1 mL/g, extraction temperature 60 °C, extraction time 3 h, twice. Under this condition, the extraction rate of SeCP was 72.91%. The optimum conditions for the enzymatic preparation of antioxidant SeCPPs were: 4% substrate concentration, 1.0% enzyme substrate ratio, 1 h digestion time, 1 kDa molecular weight retained by ultrafiltration membrane. The Se content in SeCPP was 362.378 mg/kg, and the EC₅₀ values of DPPH∙ and ABTS⁺∙ scavenging rate were 0.435 and 0.399 mg/mL, respectively. Besides, SeCPPs showed better reducing power and Fe²⁺ chelating ability.