BACKGROUND: This study examines the roles of Cyclin B1 (CCNB1), Polo-Like Kinase 1 (PLK1), and Heparanase (HPSE) in breast cancer progression using a multi-omics approach. These genes are known for their involvement in various cancer-related processes, but their precise contributions to breast cancer remain unclear. METHODS: We employed an integrative analysis combining transcriptomics, proteomics, DNA methylation profiling, immune infiltration analysis, and single-cell RNA sequencing to investigate the expression patterns, regulatory mechanisms, and functional impacts of CCNB1, PLK1, and HPSE in breast cancer. Functional assays using si-RNA knockdown of CCNB1 and PLK1 were performed to assess their roles in cell proliferation. RESULTS: CCNB1, PLK1, and HPSE are upregulated in breast tumors at the mRNA and protein levels. CCNB1 and PLK1 promote tumor growth and metastasis, while HPSE is linked to immune pathways. DNA methylation in BRCA correlates with prognosis, with PLK1 alterations protective for recurrence-free survival. High expression of these genes worsens prognosis, with CCNB1 as a risk factor for overall survival. Immune infiltration analysis associates these genes with tumor-infiltrating immune cells, highlighting HPSE's immunotherapeutic potential. Single-cell RNA sequencing confirms CCNB1 and PLK1 drive malignant proliferation and an immunosuppressive environment. Functional assays demonstrated that silencing CCNB1 and PLK1 significantly reduced breast cancer cell proliferation, indicating regulatory interactions among PLK1, CCNB1, and MKI67. CONCLUSIONS: This study provides evidence that CCNB1, PLK1, and HPSE are key players in breast cancer progression and potential biomarkers for prognosis. Furthermore, their roles in immune regulation suggest they could be promising targets for immunotherapy.