Abstract: This study used single-factor and response surface methodology to optimize the preparation technology of isothiocyanates from broccoli seeds, the factors on the yield of the isothiocyanates, such as mass ratio of enzyme to substrate, time, temperature, pH and extracted material-liquid ratio were investigated. Based on the single factor experiments, the reaction surface test was carried out by selecting the enzyme hydrolysis time, temperature and pH, and the resulting isothiocyanates were identified by the thiourea method and gas chromatography-mass spectrometry (GC-MS). The results showed that the optimum conditions were: Reaction time at 3.95 h, reaction temperature at 44.6 ℃, pH at 6.52. The yield of isothiocyanates was confirmed under the optimal conditions 8.36 mg/g (experimental) versus 8.51 mg/g (theoretical), the relative error was 1.8%. The five main compounds were identified by gas chromatography-mass spectrometry in enzymatic hydrolysis products, such as 4-(methylthio)butyronitrile, 1-butyl isothiocyanate, sulforaphane nitrile, oxazolidinethione and sulforaphane. The relative content of sulforaphane was the highest among the five compounds. In conclusion, the extraction parameters were feasible for the preparation of ITCs in broccoli seeds.