Background: Natural sulforaphane (SF) has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect. The main objective of the present work was to optimize the production of SF from broccoli seed using response surface methodology. Results: Three major factors (hydrolysis time, water volume and ethyl acetate volume) were screened out through Plackett-Burman (PB) factorial design. The methods of steepest ascent combined with central composite design (CCD) were employed for optimization of the SF production process. The optimal extraction conditions for SF production were a hydrolysis time of 13 min, a hydrolysis volume/weight ratio of 2.9:1 (v/g) and an extraction volume/weight ratio of 17.5:1 (v/g). The maximum SF yield was 14.8 ± 0.1 mg/g, a value that was in perfect agreement with the actual experimental value (14.8 mg/g). Conclusions: These results suggested that PB design combined with CCD were proved effective in screening and optimization of the parameters of SF production.