Liver hepatocellular carcinoma (LIHC) is a malignancy characterized by a high rate of recurrence, metastasis, and poor prognosis. Cytochrome b561 (CYB561) has been previously reported to be associated with tumor progression, but it has not been revealed in LIHC. The aim of this study was to investigate the prognostic value and potential function of CYB561 in LICH. The expression level, clinical correlation, prognosis, and biological function of CYB561 in LIHC were analyzed using The Cancer Genome Atlas（TCGA), Gene Expression Omnibus (GEO), TIMER2, Kaplan-Meier Plotter, and GEPIA2 databases. The expression of CYB561 in LIHC tissue samples was analyzed by immunohistochemical staining. The effect of CYB561 on the proliferation and migration of LIHC cells was investigated by using CYB561 knockdown in vitro. GSE149614 dataset was used to analyze the expression distribution of CYB561 in LIHC on a single-cell dimension. This study showed that CYB561 mRNA and protein were highly expressed in LIHC. High expression of CYB561 suggests poor prognosis in LICH patients and is an independent risk factor for LIHC. Wound-healing experiment, transwell experiment, and clonal formation experiment confirmed that CYB561 knockdown could inhibit the proliferation and migration of LIHC cells. Functional enrichment analysis showed that CYB561 was related to biological processes such as cell adhesion and immune response. KEGG enrichment analysis showed that CYB561 interacts with tumor-related signaling pathways. Single-cell analysis showed that CYB561 was mainly expressed in hepatocytes. Cells with high CYB561 expression had a higher degree of malignancy. Our study found that abnormal expression of CYB561 in LIHC suggested poor prognosis of LIHC and was related to tumor migration and proliferation. CYB561 is a potential prognostic predictor or therapeutic biomarker.© 2024. The Author(s).