Objective To explore the expression and location differences of core gene NANOG of stem cells in breast cancer stem cells (BCSCs) and non-breast cancer stem cells (Non-BCSCs) as well as the distribution of BCSCs in mammospheres of MCF-7. Methods The expression and localization of NANOG in BCSCs and NonBCSCs were detected by immunohistochemistry, immunocytochemistry, and double staining of NANOG and flourescent dye Hoechst33342. Results Immunocytochemistry and immunofluorescence staining showed that there were two types of cells in MCF-7. Most cells had big bodies and abundant cytoplasm, and NANOG mainly expressed in the cytoplasm. Other cells had small round bodies and less cytoplasm, and NANOG mainly expressed in the nucleus. NANOG and Hoechst33342 immunofluorescence double staining showed that NANOG expressed in the nucleus that could not be stained by Hoechst33342. In cells that the nucleus was stained by Hoechst33342, NANOG mainly expressed in the cytoplasm. The mammospheres were cystic sphere and hollow in center. Cells distributed along the wall of the mammosphere and the wall mainly consisted of 1 to 3 layers of cells. BCSCs mainly distributed in the inner layer of MCF-7 cystic mammospheres. Conclusion NANOG mainly express in the nucleus of BCSCs and the cytoplasm of Non-BCSCs. BCSCs mainly distribute in the inner layer of the cystic mammospheres, which provides the experimental evidence for further research of BCSCs and clinical target therapy of breast cancer.