The effects of S (as sulphate) and Se (as selenite) treatment (S mM/Se μM: 1/0, 1/50, 1/100, 1/150, 4/0, 4/50, 4/100, and 4/150) on the production of sulforaphane (an anticancer compound), the accumulation of its precursor substance, and the expression of genes related to glucoraphanin biosynthesis in broccoli were examined. Sulforaphane yield and myrosinase activity increased significantly with the combined application of 4 mM S and 100 μM Se on broccoli. Furthermore, the concentrations of glucoraphanin (a sulforaphane precursor) and methionine (a glucoraphanin substrate) slightly changed after Se application. And the strong anticancer activity of compound Se-SMC was further improved. Analysis of related gene expression showed that MY, which encodes myrosinase, was strongly induced by Se treatment. Thus, the myrosinase activity induced by Se treatment is the dominant factor affecting sulforaphane yield from glucoraphanin hydrolyzation. Selenium-sulfur biofortification provides a technical support for the cultivation of broccoli with high sulforaphane and high anti-cancer selenium compounds.