Objective:To establish a culture method of cancer stem cells(CSCs) from esophageal cancer cell lines KYSE-150 and TE-1 by serum-free medium(SFM) and identify their characteristics. Methods:KYSE-150 and TE-1 cells were cultured in serum-free medium. The cell spheres were observed and cell differentiation was induced by serum supplement. MTT assay and invasive assay were applied to examine the invasive ability of both cell spheres and their parental cells. CD44+,CD271+,CD44+CD271+ tumor spheroid cells were also detected by flow cytometry. Results:After SFM culture,KYSE-150 and TE-1 cells formed spheres,and could be passaged continuously. The differentiation of cell spheres recurred when serum was supplemented. The proliferation and invasive ability of cell spheres were higher than their parents cells. CD44+,CD271+,CD44+CD271+ in KYSE-150 cell spheres were(64.92 ± 11.04)%,(28.27 ± 6.79)%,(24.07 ± 5.39)%,which were significantly higher than those in parents cells[(37.04 ± 6.30)%,(3.69 ± 0.49)%,(1.64 ± 0.11)%,P < 0.05]. CD44+,CD271+,CD44+CD271+ TE-1 cell spheres were (6.41 ± 0.87)%,(2.58 ± 0.17)%,(0.27 ± 0.53)%,which were also significantly higher than those in parents cells[(1.87 ± 0.18)%,(0.44 ± 0.10)%,(0.09 ± 0.03)%,P < 0.05]. Conclusion:Esophageal cancer cell spheres can be isolated from KYSE-150 and TE-1 by culture with SFM. CD44 and CD271 may be the cell surface makers for these cancer stem cells.