Isothiocyanates (ITCs) are proved as one of natural anticarcinogenic compounds, which are produced from the decomposition of glucosinolates by myrosinase. The present study optimized the enzymolysis conditions (pH, addition of EDTA and ascorbic acid) for ITCs production from glucosinolates in broccoli sprouts using response surface methodology. ITCs production was clearly enhanced by a suitable pH, addition content of EDTA and ascorbic acid. The optimal enzymolysis conditions were determined to be adding EDTA 0.02 mmol and 0.16 mg ascorbic acid to 4 ml of the homogenized phosphate-citrate buffer solution (pH 4.00). ITCs profiles were identified and seven kinds of individual ITCs were detected, among which sulforaphane accounted the most. Four kinds of individual ITCs including isobutyl isothiocyanate, 4-isothiocyanato-1-butene, 1-isothiocyanato-3-methyl-butane and 1-isothiocyanato-butane are firstly reported in broccoli sprouts.