As a cofactor for mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase (MS), vitamin B12 (cobalamin, Cbl, B12) is an essential water-soluble micronutrient (MCM). Homocysteine (Hcy) and methylmalonic acid (MMA), respectively, accumulate as a result of the inactivation of MS and MCM caused by a Cbl deficiency, whether caused by dietary deficiencies or genetic faults in Cbl metabolism. The preferred blood indicators used to assess B12 status include holo-transcobalamin (holo-TC), Hcy, and MMA together with total B12 and its bioactive protein-bound form. Clinically speaking, a vitamin B12 shortage results in megaloblastic anaemia, neurological degeneration, and, if untreated, death. Subclinical vitamin B12 deficiency typically manifests without symptoms or with very mild general symptoms that are frequently misdiagnosed as unrelated illnesses. It is typically defined as a total serum B12 level of less than 200 pmol/L. The diagnostic significance of blood vitamin B12 as a standalone marker has now been demonstrated by numerous investigations. Vitamin B12 deficiency is not necessarily indicated by low serum levels, and the presence of normal or even high serum vitamin B12 levels has been linked to severe functional deficiencies of the micronutrient. This review covers the methods used to diagnosis B12 insufficiency, the utility and limitations of existing biomarkers of vitamin B12 status in newborn screening, infant and adult diagnostics, and atypical discoveries of vitamin B12 status in a variety of human illnesses.