Abstract: To investigated the effects of spray application of zinc sulfate (ZnSO₄) solution on the accumulation of crucial bioactive components and antioxidant activity, and explored the regulatory mechanism of zinc sulfate on glucosinolates in broccoli flower buds and seedlings at the molecular level, seven-day-old broccoli sprouts were treated with one of four concentrations (1, 2, 4, and 8 mmol/L) of aqueous ZnSO₄ solutions or water (0 mmol/L ZnSO₄ as the control). The effects of each ZnSO₄ treatment on the bioactive components of broccoli sprouts were investigated by determining the contents of chlorophyll, ascorbic acid, total flavonoids, total phenols, and total glucosinolates, as well as myrosinase activity and DPPH free-radical scavenging rate. The expression levels of glucosinolate synthesis structural genes in the sprouts were analyzed using a real-time quantitative PCR detection system. The results showed that the chlorophyll content of all ZnSO₄ treatments was lower than that of the control. The ascorbic acid content tended to be elevated with an increase in ZnSO₄ concentration and was higher than that of the control for all ZnSO₄ treatments. The total polyphenol content was the highest in response to 8 mmol/L ZnSO₄ treatment and was significantly (P<0.05) higher than that of the control. The contents of total flavonoid content, total glucosinolates, glucoraphanin, myrosinase activity and maximum DPPH free-radical scavenging rate were greatest after treatment with 2 mmol/L ZnSO₄ solution. The expression levels of CYP83A1, CYP79F1, and FMO-GSOX2 were significantly (P<0.05) increased in response to 2 mmol/L ZnSO₄ treatment. Thus, spray application of exogenous ZnSO₄ promoted the expression of crucial genes involved in thioredoxin synthesis. FMO-GSOX2 showed highly significant (P<0.01) positive correlation with total glucosinolates, RAA, aliphatic glucosinolates, and myrosinase activity. CYP79F1 and CYP83A1 showed significant (P<0.05) positive correlation with total glucosinolates, RAA, and aliphatic glucosinolates. It indicated that the up-regulation of FMO-GSOX2, CYP79F1 and CYP83A1 expression promoted the synthesis of total glucosinolates, aliphatic glucosinolates and RAA. In conclusion, the results of this study showed that spray application of an appropriate concentration of exogenous ZnSO₄ promoted up-regulation of the expression of critical glucosinolate synthesis-related genes, and that 2 mmol/L ZnSO₄ solution effectively increased the contents of bioactive compounds and antioxidant activity in broccoli sprouts.