Abstract: OBJECTIVE To prepare chlorambucil liposome, and to optimize the formulation of chlorambucil liposome. METHODS Chlorambucil liposome were prepared by film-ultrasound method and the encapsulation efficiency was measured by microcolumn centrifugation-HPLC method. The single factors such as the ratio of cholesterol to phospholipid, the ratio of drug to phospholipid, the pH value of aqueous phase and the concentration of phospholipid were studied. And the orthogonal design was adopted to obtain the optimized prescription, and the quality evaluation was carried out. RESULTS The best prescription was as follows:the ratio of cholesterol to phospholipid was 1:3; the ratio of drug to phospholipid was 1:10; the pH value of aqueous phase was 7.4 and the concentration of phospholipid was 0.3%. The encapsulation efficiency of the chlorambucil liposome prepared according to the prescription was >87%, the average particle size was 84.71 nm, and the PDI was 0.167. CONCLUSION The chlorambucil liposome prepared according to the optimized formulation has higher encapsulation efficiency with small and uniform particle size.