Abstract: To prepare bioactive peptides from rapeseed cake directly, the superfine grinding technology was used, and the resulting rapeseed cake superfine powder was used as the material. Single-factor experiments and response surface analysis were used to optimize the preparation extraction. The active peptides were purified by membrane separation and Sephadex G-15 membrane separation, and their antioxidant activity and structure were evaluated. The results showed that the optimal preparation conditions were as follows: grinding time 30 min, solid-to-liquid ratio 1:20(g/mL), pH2.94, enzyme dosage 6300 U/g, temperature 55 ℃, and enzymatic hydrolysis time 2.25 h. Under these conditions, the yield of peptides could reach 83.28%. The F-2 fraction from hydrolysates with molecular mass less than 1000 Da showed good antioxidant activity, with an ABTS⁺ free radical eliminating rate of 71.62% at a concentration of 1 mg/mL. Furthermore, five peptides were identified, namely PF, SF, TF, IAIPF, and PLDLL which were predicted to have much higher activity. Overall, the results of this study can provide a theoretical basis for the exploration of bioactive peptides directly from rapeseed cakes.