Abstract: Nisin is a natural antimicrobial agent. To enhance the stability of Nisin, this study selected soybean phospholipids and phytosterol butyrate ester as membrane materials, and prepared complex liposomes co-encapsulating Nisin and phytosterol butyrate ester via the film-ultrasound dispersion method. The encapsulation efficiency was selected as the evaluation criterion. Systematic single-factor experiments were conducted to investigate four key parameters: the mass ratio of Nisin to phospholipids, the mass ratio of phytosterol butyrate ester to phospholipids, the pH value of phosphate buffer solution, and ultrasonic time. The physicochemical properties of the complex liposomes were characterized using multiple techniques. Dynamic light scattering (DLS) was employed to determine particle size distribution, while electrophoretic light scattering was used to measure Zeta potential. The encapsulation efficiency was analyzed, and morphological examination was performed using transmission electron microscopy (TEM). Fourier-transform infrared spectroscopy (FT-IR) was employed to analyze molecular interactions; X-ray diffraction (XRD) was used to characterize the crystalline structure, and differential scanning calorimetry (DSC) was applied to assess thermodynamic properties. The results indicated that the optimum conditions were as follows: the mass ratio of Nisin to phospholipids was 1 ∶ 4, the mass ratio of phytosterol butyrate ester to phospholipids was 1 ∶ 20, the pH value of phosphate buffer was 5.0, and ultrasonic time was 4 min. Under the optimal conditions, the encapsulation efficiency of complex liposomes was (85.44 ± 1.62)%, the particle size was (109.3 ± 9.0) nm, the polydispersity index (PDI) was 0.338±0.012, and the zeta potential was ( -14.78±1.38) mV. Results from FT-IR, XRD, and DSC confirmed the successful encapsulation of Nisin and phytosterol butyrate ester within the complex liposomes. Transmission electron microscopy ( TEM) analysis revealed that the complex liposomes were uniformly dispersed spherical vesicles with regular morphology. This study provides scientific evidence and novel insights for expanding the practical applications of Nisin in food preservation and freshness maintenance.