Mycobacterium tuberculosis, responsible for tuberculosis (TB), remains a major health problem worldwide and is one of the infectious diseases causing increased morbidity and mortality worldwide. Biotin, namely vitamin H, is an important cofactor necessary for fatty acid biosynthesis, gluconeogenesis and amino acid metabolism in organisms including Mycobacterium tuberculosis. Due to its inability to ingestion biotin from outside, Mycobacterium tuberculosis can only obtain biotin through biotin biosynthesis. Different from the classical BioC-BioH, BioI-BioW and non-classical BioZ pathways, Mycobacterium tuberculosis synthesized biotin by "BioC-BioH(2)" pathway in the early stage. This review focuses on the unique biotin synthesis pathway of Mycobacterium tuberculosis and its key genes, especially the response of this pathway and biotin-dependent carboxylase to tuberculosis first-and second-line drugs, as well as inhibitors and natural products targeting biotin synthesis.