Objective To optimize preparation of freeze-dried 16-dehydropregnenolone (16-DHP) liposome and evalu-ate the quality. Methods The freeze-dried liposome was prepared by rotary-evaporated film-ultrasonication with lyophilization. Single factor experiments and orthogonal design were used to optimize the prescription and preparation,and the quality was evalua-ted. Results The optimum preparation technics were as follows: the concentration of PC-98T was 6. 5% (W/ V), the quantity ratio of PC-98T to cholesteryl sodium sulfate salt was 4∶1,hydration temperature was 60 ℃ , ultrasonic time was 2 min,and the cryoprotectant was 7. 5% glucose. The average particle size, Zeta potential and encapsulation efficiency were (121. 3±48. 7) nm,-41. 9 mV and (98. 7±0. 1)% , respectively. Conclusion The formulation of freeze-dried 16-DHP liposome is reasonable, the technology is practicable and stable.