In order to improve the utilization efficiency of aquatic products and reduce the environmental pollution caused by waste protein, this paper explored the retrieving and using of Spanish mackerel processing wastes that are rich in protein, thus to search for the optimum conditions for preparing antioxidant peptides from Spanish mackerel, reduce the production cost and improve the economic benefit. Using waste proteins from Spanish mackerel production as substrate and bacillus thuringiensis Hy-4 as fermentation strain, Design-Expert software was applied to optimize the conditions for antioxidant peptides production. On the basis of single factor experiments, Plackett-Burman design was utilized to evaluate the effects of six variables influencing antioxidant peptides preparation from Spanish mackerel, including fermentation time, fermentation temperature, initial pH, inoculum quantity, ratio of material to solvent and cell age.  Among the six variables studied, fermentation temperature, initial pH and ratio of material to solvent had significant effects on the total antioxidant activity of fermentation solution (P<0.05). Steepest ascent method was used to approach the optimal region of the total antioxidant activity. The regression analysis was further carried out using Box-Behnken design and response surface analysis. By solving the regression equation, the highest antioxidant peptides production was obtained at fermentation time 48h, fermentation temperature 30℃，initial pH 6.8, inoculum quantity 2%, material-to-solvent ratio 2.9%, and cell age 24h. The total antioxidant activity of fermentation solution reached to 537.73U, increased by 57.2% compared to the initial 342.02U. Compared with the predicted value of regression equation 570.11U, the relative error is 5.7%, illustrating that the model can better predict the total antioxidant activity of fermentation solution fermented by Hy-4.