Abstract: In order to investigate the optimal extraction process of sulforaphane from broccoli seeds and evaluate its immunomodulatory activity, broccoli seeds as the raw material, the effects of parameters including solid-liquid ratio, ethanol concentration, extraction time, extraction temperature and extraction times on the yield of sulforaphane were examined. The parameters were optimized by orthogonal experimental design. The immunomodulatory activity of its extract contains sulforaphane was explored based on the immunodeficiency zebrafish model. The results indicated that the highest yield of sulforaphane was 18.24 mg/g under the conditions of a 1:22 g/mL solid-liquid ratio, ethanol concentration of 10%, 1 h of extraction time, 40 ℃ of extraction temperature and two extraction circles. The immunomodulatory activity tests showed that the extract contains sulforaphane had strong immunomodulatory activity and could significantly enhanced macrophage phagocytosis in zebrafish at high dosage (200 μg/mL) and medium dosage (100 μg/mL), improve T cell reduction at medium dosage (100 μg/mL) and low dosage (50 μg/mL), and improve neutropenia at high dosage (200 μg/mL), medium dosage (100 μg/mL) and low dosage (50 μg/mL). By optimizing the extraction process of sulforaphane from broccoli seeds and evaluating its immunomodulatory activity, the study provided a theoretical and practical basis for the research and application of the extract contains sulforaphane.