Breast cancer is the second leading cause of cancer–related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome In spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression Differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next Generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p