Abstract: In order to maximize the utility of giant salamander products and to explore the sustainable and efficient bacteriostatic agents, antimicrobial peptide from giant salamander (Andrias davidianus) skin (APGSS) was prepared by ultrasound-assisted enzymatic hydrolysis, with the extraction process optimizing by response surface methodology. The extracted peptide was preliminarily purified, and subsequently characterized for its structural characteristics. Its bacteriostatic mechanism was investigated by techniques including infrared chromatography, circular dichroism, time-flight mass spectrometry, liquid chromatography-tandem mass spectrometry. The results showed that the optimized extraction conditions for APGSS were determined as follows: 16.6 minutes ultrasound duration, 120 W ultrasound power, 18000 U/g enzyme addition, 5.85 h enzyme digestion time, and 21.3% substrate concentration, the verification test measured the yield of APGSS was 1.164%±0.021%. Infrared spectroscopy and circular dichroism analysis revealed the secondary structure proportion of APGSS: 45.54%±1.59% random coil, 22.67%±0.78% α-helix, 18.42%±1.05% β-sheet and 13.41%±0.6% β-turn. Mass spectrometry showed APGSS had a low molecular weight and a high content of hydrophobic amino acids. Bacterial respiration inhibition test indicated that APGSS exerts antimicrobial effects by inhibiting the Embden-Meyerhof-Parnas pathway in Staphylococcus aureus. Electron microscopy further demonstrated that APGSS compromises the bacterial cell membrane integrity of S. aureus and change its permeability. These findings provide valuable insights for the sustainable utilization of giant salamander resources and the development of safe, efficient natural food preservatives.