Cholangiocarcinoma (CCA) is a malignant tumor arising from the epithelial cells of the biliary tract. It is characterized by high heterogeneity, frequent late diagnosis, high rates of metastasis, and overall poor prognosis. Unfortunately, the development of sensitive biomarkers and effective therapeutic approaches remains challenging and an unmet need. In recent years, advances in molecular biological techniques have significantly enhanced our understanding of the molecular mechanisms underlying CCA pathogenesis. This review summarized the biomarkers identified in CCA, highlighted the critical genes and signal transduction pathways closely associated with tumor cell proliferation and migration, inflammation regulation, angiogenesis, and tumor microenvironment, and discussed the complex regulatory roles of epigenetic alterations in CCA initiation and progression. These recent insights not only deepen our understanding of the molecular complexity of CCA but also lay a solid foundation for the development of precision medicine and individualized therapeutic strategies for patients with this aggressive malignancy.