Objective · To improve the purity and survival rate and to reduce the time for sorting SP subpopulation of human lung cancer cell A549 by optimizing flow- cytometry sorting method. Methods · Single cell suspension was prepared, incubated with different concentration of Verapamil, and stained with different concentrations of Hoechst33342. The ratio of SP cells was detected following PI marking. One time sorting method (purity mode) and two time sorting method (enrichment and purity mode) were used to sort SP and MP cells with the best staining concentration, 100 μm nozzle, and sorting velocity of 10 μL/min. The sorting purity and time were compared between two methods. SP cells and MP cells sorted with the two time sorting method underwent tumor forming experiment and confocal observation after Lyso-Tracker Red and Hoechst 33342 stainings. Results · The purity of SP cells sorted with two times sorting method was more than 99% and the sorting time was less than 5.2 h for sorting 1×108 cells. Under confocal microscopy, the size of SP cells was not different from that of MP cells and MP cells had more lysosomal granules than SP cells. Both cells were able to be stained with Hoechst 33342 and the cell survival rate was not affected. In mice tumor forming experiment, the size of tumor formed with SP cells was significantly larger than that formed with MP cells. Conclusion · The optimal staining concentration of Hoechst33342 for A549 lung cancer cells is 6 μg/mL. Using two time sorting method and FACSAria Ⅱ flow sorter to sort SP cells can not only significantly improve the purity of SP cells, but also greatly reduce the sorting time without affecting the survival rate of SP cells.