Stem-like cancer cells are called cancer stem cells (CSCs) or tumor stem cells (TSCs). Methods for sorting CSCs are mainly based on the marker (CD133+/CD44+) or side population cells. However, CD133+/CD44+ cells or side population cells are very rare or even undetectable. In the present study, the tumor sphere of human gastric cancer (HGC) cell line HGC-27 was used for CSCs enrichment, and stem-like characteristics were verified by Hoechst 33342 staining technology, cell growth rate assays, sphere differentiation assay, clone formation, chemotherapy resistance study and tumor formation in an animal model. Our results demonstrated that the tumor sphere cells of HGC-27 cell line could be used to enrich CSCs, which may contribute to human gastric cancer stem cell biology research.