Vitamin B12 is an essential water-soluble vitamin required for critical biological processes such as DNA synthesis, erythrocyte maturation, and maintenance of nervous system integrity. Deficiency of vitamin B12 can lead to serious clinical outcomes, including megaloblastic anemia, and potentially irreversible neurological damage. Conversely, hypercobalaminemia may be associated with severe disorders, including solid neoplasms, hematological malignancies and, in some cases, may result from inappropriate supplementation or immunoglobulin–B12 macro-complexes. Although current guidelines recommend total serum vitamin B12 and holotranscobalamin (holoTC) as first-line biomarkers, total serum vitamin B12 remains the most widely used test in routine clinical practice. However, since holoTC represents the biologically active fraction of vitamin B12 available for receptor-mediated cellular uptake, it appears to provide a more reliable assessment of cobalamin status, particularly in specific clinical contexts. Compared with total vitamin B12 measurement, holoTC is assessed using a more limited number of analytical methods and the majority of available kit are aligned with the WHO reference standard, thereby improving inter-assay harmonization. This review explores literature data about the role of vitamin B12 and holoTC, discussing analytical challenges and clinical interpretation, highlights the potential advantages of holoTC over total serum B12.