Objective:To investigate the content of cancer stem-like cells in different human breast cancer cell lines and to explore the methods for enriching cancer stem-like cells in MCF-7. Methods:The percentage of SP cells and CD44+CD24-/low cells in human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435s were detected with flow cytometry. MCF-7 was separated by Percoll gradient centrifugation into five subpopulations. In order to screen which subpopulation contained the highest content of SP cells, each subpopulation was detected with flow cytometry. Then MCF-7 was cultured in serum-free DMEM/F12 containing EGF, bFGF, BSA and insulin. The percentage of CD44+CD24-/low cells was detected with flow cytometry after 1,2,3weeks,respectively. Results:The percentage of SP cells was 3.61% in MCF-7 and 2.72% in MDA-MB-435s. There was no SP cells detected in MDA-MB-231. The percentage of CD44+CD24-/low cells was 0.87% in MCF-7, 0.59% in MDA-MB-231 and 94.04% in MDA-MB-435s. After the separation of Percoll gradient centrifugation, subpopulation D of MCF-7 was rich in SP cells,and reached up to 25.23%. The cancer stem-like cells in MCF-7 grew as clonal nonadherent spherical clusters. CD44+CD24-/low cells in MCF-7 increased after culture in serum-free medium. The percentage of CD44+CD24-/low cancer stem-like cells reached up to 29.23% after 3 weeks culture in serum-free medium. Conclusion:SP cells detection and CD44+CD24-/low cells detection are two commonly used methods in detecting breast cancer stem cells. But the results of two methods often disagree. Each method has its own limitation. The cancer stem-like cells in MCF-7 can enrich after separation by Percoll gradient centrifugation or culture in serum-free medium.