Objective To prepare cationic Vancomycin hydrochloride multivesicular liposome (MVL) and to inspect its quality. Methods Cationic Vancomycin hydrochloride MVLs were prepared by double emulsion method, and the storing solution of Vancomycin was prepared. The analysis method of Vancomycin in vitro was established; the specificity, precision, and resorption rate were estimated. Reverse phase high performance liquid chromatography (RP-HPLC) was used to determine the concentration of Vancomycin, encapsulation efficiency, and release characteristics in vitro. The formulation and pharmaceutical process were optimized by single factor experiments and orthogonal experimental design with the factor of encapsulation efficiency as the criteria. The liposome morphology was observed by optical microscopy and transmission electron microscopy. The particle size and Zeta potential were determined by Malvern instrument. The stability was analyzed by dynamic analysis. Results An RP-HPLC method was established for the assay of Vancomycin. The analysis method was precise, simple, and reliable for the quality control of Vancomycin. Vancomycin hydrochloride MVLs were round and well-distributed. The average particle size and the encapsulation efficiency were 3.3 μm and 24.9%, respectively. Zeta potential was 24.53 mV, and 90.5% of Vancomycin hydrochloride was released after 264 hours in normal saline under 37℃. Cationic Vancomycin MVLs were stored for 1 month at 4 ℃, which mantained good stability. Conclusion Cationic Vancomycin hydrochloride MVLs have good appearance, high encapsulation efficiency, good stability, and significant sustained release properties.