Purpose To identify whether serum-free culture can enrich breast cancer stem cells from MCF-7 human breast cancer cell line. Methods MCF-7 human breast cancer cell line by serum-free culture and serum culture technology were cultured, its cell mor-phology and growth pattern were observed by the inverted microscope. The expression of stem cell surface molecular makers CD24, CD44 was observed by the inverted fluorescence microscope and the flow cytometry, the proportion of different subpopulation cells was detected by the flow cytometry. At the same time, difference of the cell cycle was detected by the flow cytometry. Results The cell line cultured by serum-free culture grew in the form of suspended microspheres of different sizes in the medium, but the cell line cul-tured by serum culture grew in the form of monolayer adherent growth. There was no obvious difference in the expression of stem cell surface molecular makers CD44 between the suspended microsphere cells and the adherent cells, but the expression of CD24 in the sus-pended microsphere cells decreased compared to the adherent cells. The proportion of CD44 + /CD24 -/low phenotype cells in the suspen-ded microsphere cells and the adherent cells was (86. 93 ± 0. 53)% and (19. 98 ± 0. 62)%, respectively (P<0. 05), the proportion of CD44 + /CD24 + phenotype cells was (12. 68 ± 0. 59)% and (79. 90 ± 0. 57)%, respectively (P<0. 05). The proportion of mitotic cells in the suspended microsphere cells and the adherent cells was ( 18. 85 ± 2. 26 )% and ( 43. 91 ± 1. 81 )%, respectively ( P<0. 05), the proportion of quiescent cells was (64. 92 ± 2. 07)% and (39. 82 ± 1. 77)%, respectively (P<0. 05). Conclusion CD44 + /CD24 -/low breast cancer stem cells can be effectively enriched by the serum-free culture technology.