Abstract: Cancer stem cells (CSCs) stand for a small population of cancer cells that have the potential of self-renewal and differentiation and lead to strong chemotherapy resistance,tumor recurrence and metastasis.Establishing a stable model for the study of CSC drug resistance is necessary.Herein a culture approach of cancer stem cells was developed by using serum-free suspension culture system,and MCF-7 breast cancer cell spheroids were formed by using the method.Cancer stem cells in these spheroids were identified via alkaline phosphatase staining,flow cytometer analysis,quantitative Real-time PCR and Western Blot.Subsequently,doxorubicin,a common used chemotherapeutic drug,was applied to these CSCs,which showed lower drug uptake efficiency and high level of resistance compared to normal cultured MCF-7 cells.Further study showed that the membrane transporting protein ABCG2,was highly expressed in CSCs,which also related to the chemotherapeutic drug resistance.