Abstract: To address the issues of poor stability and degradation of antimicrobial peptides in food preservation and pharmaceutical applications, liposome encapsulation technology was employed to enhance the stability and bioavailability of antimicrobial peptide W1. The antimicrobial peptide W1 was extracted and purified from Bacillus subtilis SNBS-3 using ammonium sulfate precipitation, Sephadex-G25 gel chromatography, and high-performance liquid chromatography (HPLC). Its molecular sequence was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Subsequently, W1-loaded liposomes were prepared using the film dispersion method, and optimal preparation conditions were determined through single-factor experiments and response surface methodology. The physicochemical properties of the liposomes were evaluated through particle size, Zeta potential, encapsulation efficiency, and microscopic structural analysis. The stability and antibacterial activity of the liposomes were tested under different temperature, pH, and enzyme treatment conditions. Additionally, cell experiments assessed the cytotoxicity of W1 liposomes on human liver cells (LO2). The results indicated that a highly active fraction was obtained by Sephadex-G25 purification, further purified by HPLC to obtain the core antibacterial peak, and the antimicrobial peptide W1 sequence was identified as IGLFGGAGVGK by LC-MS/MS. The optimal preparation conditions were determined to be a mass ratio of soybean phosphatidylethanolamine (SPE) to β-sitosterol of 5.94:1, a mass ratio of SPE to W1 of 5.18:1, and an ultrasonication time of 6.75 minutes. Under these conditions, W1-loaded liposomes were obtained with a particle size of 105.88±6.35 nm and an encapsulation efficiency of 68.2%±1.5%. Good physicochemical stability was exhibited by the resulting liposomes. Antibacterial tests revealed that the minimum inhibitory concentration (MIC) of W1 liposomes against Staphylococcus aureus was 32 mg/mL. Cytotoxicity assays demonstrated that W1 liposomes maintained LO2 cell viability above 90% at concentrations below 512 mg/mL (P>0.05), indicating favorable biocompatibility. These findings suggest that liposomal encapsulation significantly enhances the stability and antimicrobial efficacy of W1, providing a promising strategy for its application in the food and pharmaceutical industries.