Cyclin B1 (CCNB1) encodes a regulatory protein essential for the regulation of cell mitosis, particularly in controlling the G2/M transition phase of the cell cycle. Current research has implicated CCNB1 in the progression of various types of cancer, including gastric cancer, breast cancer, and non-small cell lung cancer. In this study, we conducted a pan-cancer analysis of CCNB1 to investigate its prognostic significance and immunological aspects. Our comprehensive investigation covered a wide range of analyses, including gene expression, promoter methylation, genetic alterations, immune infiltration, immune regulators, and enrichment studies. We utilized multiple databases and tools for this purpose, such as The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, the Human Protein Atlas (HPA), the University of Alabama at Birmingham CANcer data analysis Portal (UALCAN), the Gene Expression Profiling Interactive Analysis (GEPIA), the DNA Methylation Interactive Visualization Database (DNMIVD), the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), Sangerbox, and cBioPortal. Data analyses were executed using GraphPad Prism software, R software, and various online tools. Our findings demonstrated a significant increase in CCNB1 expression across 28 cancer types. Elevated CCNB1 expression correlated with decreased overall survival (OS) in 11 cancer types and disease-free survival (DFS) in 12 cancer types. Additionally, DNA promoter methylation levels were significantly decreased in 14 cancer types. Furthermore, the study verified a significant association between CCNB1 expression and immune infiltration, immune modulators, microsatellite instability (MSI), and tumor mutational burden (TMB). Enrichment analysis indicated that CCNB1 is involved in multiple cellular pathways. Collectively, our results suggested that CCNB1 has the potential to serve as a valuable prognostic biomarker and may be a promising target for immunotherapy in various cancer types.