Abstract: The objective of this study was to investigate the optimal preparation process and digestive properties of sulforaphane from Raphanus sativus seeds prepared by enzymatic method. Single factor experiment and response surface methodology were employed to optimize the enzymolysis conditions for extracting sulforaphane from red Raphanus sativus seeds, with sulforaphane yield as index. Additionally, an in vitro simulated gastrointestinal digestion model was used to evaluate the changes in content and antioxidant activity of sulforaphane in ethyl acetate extracts of Raphanus sativus seeds. The results revealed that the optimal preparation conditions were as follows: The enzymolysis time was 22 minutes, the enzymolysis temperature was 40 ℃, the vitamin C concentration was 0.8 mg/g. Under these conditions, the sulforaphane yield was 2.11±0.02 mg/g, which closely matched the predicted value of 2.14 mg/g. The relative error for the enzymolysis process was 1.4%, demonstrating its feasibility. Furthermore, in vitro digestion simulation showed that the retention rate of sulforaphane was 79.64% and 76.22% in gastric and intestinal fluids, respectively. The free radical scavenging rates of ABTS⁺, DPPH and OH in gastric fluid reached 54.23%, 33.90% and 15.07%, respectively, and all the values had significant (P<0.05) reductions in comparison with that of the undigested extracts. However, no significant (P>0.05) difference in the three radical scavenging rates was observed in intestinal and gastric digestive fluids. This study enriched the research on the preparation of sulforaphane by enzymolysis method, and provided experimental basis for the development and high value-added utilization of Raphanus sativus seeds.