Abstract：NADH-dependent oxidoreductases are valuable tools for the biological synthesis of fine chemicals and chiral compounds. As the reducing equivalent, cofactor NADH plays a critical role in those reactions. Due to the high cost of the NADH cofactors, in situ NADH regeneration is required for preparative applications. NAD+-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energy supply of methylotrophic microorganisms. FDH captures increasing attention in recent years, and is widely used in enzymatic syntheses of chemical compounds as a versatile biocatalyst for NADH regeneration consumed in the main reactions. This review covers the latest developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for NADH regeneration from the following aspects: chemical stability, thermal stability and cost. Future development of FDH used as NADH regeneration platform is also illustrated.