More sensitive and effective diagnostic markers for the detection of breast cancer are urgently needed. The microRNA-183/182/96 cluster has been reported to be involved in tumorigenesis and progression in a variety of cancers, and it is a promising cancer prognostic biomarker. The goal of this study was to determine the expression levels of the miR-183/182/96 cluster in breast cancer tissues and evaluate its prognostic role in breast cancer. Real-time quantitative polymerase chain reaction analysis (qRT-PCR) was used to detect the expression levels of the miR-183/182/96 cluster in 41 breast cancer tissues and adjacent normal tissues (control tissues) and also in different mammary cell lines. In situ hybridization (ISH) of the miR-183/182/96 cluster on 131 tissue microarrays (TMAs) was used to statistically analyze its prognostic role. The miR-183/182/96 cluster levels were significantly higher in breast cancer tissues than in control tissues. The miR-183/182/96 cluster was also upregulated in human breast cancer cell lines. An increased miR-183/182/96 cluster level was correlated with local relapse, distant metastasis and poor clinical outcomes. Our findings improve our understanding of the expression level of the miR-183/182/96 cluster in breast cancer and clarify the role of the miR-183/182/96 cluster as a novel prognostic biomarker for breast cancer.