Abstract: Nobiliside A (Nob A) liposomes were prepared. Its assay method of content and encapsulation efficiency (EE) were established, and hemolytic activity with Nob A solution in vivo and in vitro were compared. Preparative method, phospholipid content, ratio of phospholipid to cholesterol and ratio of drug to lipids were optimized by single factor exploration. According to the optimized results, 3 batches of Nob A liposomes were prepared, then high performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) method was used to determine the content of Nob A and minicolumn centrifugation method to determine EE, transmission electron microscope was used to detect the morphology and laser scatter analysis to evaluate particle sizes of the liposomes. The hemolytic activity was studied both in vivo and in vitro. The results indicated that HPLC-ELSD method and minicolumn centrifugation method used in this study are simple, applicable and accurate for the determination of the content and EE of Nob A liposome respectively .Nob A liposomes have a high EE with spherical shape and uniform size by using the film ultrasonication technique. When the ratio of phospholipid to cholesterol was 2∶1 and the ratio of Nob A to lipids was 1∶40, the mean EE of Nob A liposomes was 95.7% and the mean diameter was 87.6 nm. Liposomes inhibited the hemolytic activity of Nob A in vivo and in vitro sharply. As for its low hemolytic activity in vivo and in vitro, Nob A liposomes are optimistic to be used by intravenous injection.