The use of ultrasonic-assisted acetate extraction to improve the efficiency of the conventional acetate extraction technique with the aim of extracting antimicrobial peptides from Mytilus edulis by-product using acetate solution was investigated. Based on the single factor experiment, response surface methodology was used to optimize experimental conditions for extraction of antimicrobial peptides. The optimum extraction conditions for maximum antimicrobial peptide yield were established to be ultrasonic power of 200 W, acetate concentration of 0.8%, liquid-material ratio of 6∶1 (mL/g), and ultrasonic extraction time of 16 min. Under abovementioned conditions, the yield and the zone of inhibition against Staphylococcus aureus were 1.55% and 7.52 mm, respectively. The antimicrobial peptides yield and the relative of inhibition zones were significantly higher than those of the traditional acetic acid extraction method (P＜0.05). The optimized technology was feasible and the antimicrobial peptides had antibacterial activity.