Abstract: Prickly ash seed protein was hydrolyzed by pepsin for antibacterial peptides preparation. One-factor-at-a-time method and response surface analysis based on Box-Behnken design, using four factors and three levels of response surface methodology to analyzed each factor. The inhibitory rate on Escherichia coli in response to the value of establishing mathematical model and the optimal hydrolysis condition was substrate concentration 4.9%, enzyme/substrate ratio (g/g) 0.9∶100, pH2.0, hydrolysis temperature 32℃ and hydrolysis time 3h. Under these conditions, the obtained hydrolysate showed an inhibitory rate as high as60.96%.