Abstract: With the mustard seeds as material and the oil rate as indicator,the optimum enzyme was determined first. On the basis of investigating the effect of enzymatic hydrolysis temperature,enzymatic hydrolysis time and ratio of material to liquid on the oil yield of mustard seed,the aqueous enzymatic extraction process was established based on these combined with the response surface methodology optimization method,and the components of mustard seed oil was analyzed. The results showed that alkaline protease had the best effect on the oil yield of mustard seeds. The optimization conditions were as follows:2.5% enzyme(g/100 mL),pH10,enzymatic hydrolysis temperature 45 ℃,7:1 (mL/g)liquid-solid ratio and enzymatic hydrolysis time 6 h,the oil yield of mustard seeds can reached 23%. The real value was 1.8% higher than the predicted value. Analysis of fatty acid components by GC-MS showed that the relative content of unsaturated fatty acids,saturated fatty acids,erucic acid,eicosenoic acid and the total content of oleic acid were 81.34%,12.40%,13.51%,16.42%,and 50.72% respectively. The results could provide a new method for the comprehensive utilization of mustard seed oil in future.