Abstract: OBJECTIVE To optimize conjugated estrogen liposome thermosensitive gel for vaginal drug delivery and evaluate its properties. METHODS Box-Behnken design and response surface methodology was used to optimize the formulation of liposome. The thermosensitive gel was prepared by cold solution method. Franz diffusion cell was employed for the in vitro release study. Shear rate scanning, temperature scanning and frequency scanning were performed by the rheometer. Vaginal retention was investigated by florescence imaging. RESULTS The optimal formulation contained 0.030 0 g cholesterol and 0.123 6 g lecithin(1.00:4.12), 0.021 6 g API and 0.153 6 g lipid(1.00:7.11), 3.67 mL PBS as hydrated medium. The entrapment efficiency was(93.99±0.92)% and the gelatination temperature was(33.0±0.5)℃. The in vitro accumulative release rates of liposome thermosensitive gel within 48 h was (77.82±0.75)%, the curve was well fitted with the Higuchi model. When the gelation temperature was reached, rheological properties changed significantly. IR820-labeled liposome thermosensitive gel showed high fluorescence intensity in vagina of KM mice and the intensity decreased slowly. CONCLUSION The formulation and preparation process of conjugated estrogen liposome thermosensitive gel are reasonable. It can transform into semisolid at gelation temperature and prolong retention in vagina.