Objective：To sort the CD133+/CD44+ stem cell through magnetic bead cell sorting（MACS） from human prostate cancer cell line PC-3 and to lay the foundation for the further functional research. Methods：Expressions of CD133 and CD44 on the membrane of PC-3 cell before and after MACS were detected through flow cytometry（FCM）. Formation of spheres cultured by serum-free medi－um（SFM） was observed and subsequent immunofluorescence（IF） assay was made. Differences in the morphology and proliferation capacity of PC-3 cell before and after MACS were distinguished. Expressions of prostatic acid phosphatase（PAP） before and after the differentiation assay were detected by immunohistochemistry（IHC） and Western blot. Results：Positive expressions of CD133 and CD44 of PC-3 cell were （1.33±0.05）% and （0.87±0.06）% through FCM，while those after MACS were （84.82±0.07）% and （99.91±0.03）% respectively. Positive expressions of CD133 and CD44 remained after SFM detected by IF. Proliferation capacity was higher in CD133+/CD44+ cell than in PC-3 cell（t=11，P=0.008） and CD133+/CD44+ cell after inducement（t=40.1，P=0.001）. Ex－pressions of PAP of CD133+/CD44+ cell were positive after being induced by transforming growth factor-β and were negative in the uninduced CD133+/CD44+ cell based on IHC and Western blot. Conclusions：CD133+/CD44+ cell had some characteristics of stem cell after MACS from PC-3 cell line through initial functional identification，which could be matting for further explore of the prostate cancer stem cell.