Abstract: Enzymatic hydrolysis conditions of Skipjack tuna dark flesh by Alcalase were investigated in this paper.Taking initial p H, enzyme dosage, hydrolysis temperature, hydrolysis time as factors and reducing power of hydrolysate as response, the enzymatic hydrolysis conditions of dark flesh were optimized based on single factor experiments. It turned out that the optimum hydrolytic conditions were Alcalase with a p H9.9, dosage of5183U/g, temperature 49℃ and hydrolysis for 4.75 h, and result in a reducing power of 1.4308/OD700 nmwhich was close to the predicted value 1.4393/OD700 nm. The parameters of skipjack dark flesh hydrolysate process by response surface methodology were more reliable, providing technique support for development and utilization of Skipjack dark flesh.