Abstract: The accumulation of isothiocyanate in germinated broccoli under ZnSO₄-melatonin treatment was optimized. The isothiocyanate content, bud length and fresh weight of broccoli during germination were used as indices to study the effects of germination temperature, germination time, ZnSO₄ and melatonin treatment concentration on isothiocyanate by single factor experiment first, and on this basis, it was further optimized by response surface experiment. Meanwhile, the changes of isothiocyanate metabolism related indexes were analyzed. The results showed that germination temperature, germination time, ZnSO₄ and melatonin treatment concentration all had significant effects on isothiocyanate enrichment. Response surface optimization experiment showed that when the broccoli was treated with 4.69 mmol/L ZnSO₄ combined with 13.18 μmol/L melatonin at 30 ℃ for 3 days, the isothiocyanate content was up to the highest, which was 451.27 mg/100 g m_f. The contents of glucosinolates and sulforapenin and the activity of myrosinase in broccoli sprouts under ZnSO₄ combined with melatonin treatment were significantly higher than those in the control group(P<0.05). This study showed that ZnSO₄-melatonin treatment was an effective means to enrich isothiocyanate in broccoli sprouts. The optimized germination process can provide technical support for the production of broccoli sprouts rich in isothiocyanate.