Abstract: Hydrogen is a clean fuel with high combustion value. With carbohydrates and water as the substrate by joint catalysis of 13 enzymes, the products of both hydrogen and carbon dioxide were generated using the processes of cell-free synthetic enzymatic pathway biotransformation (SyPaB). In theory, twelve mols of hydrogen was produced by consumption of one mol glucose, which was triple in comparison with that by the fermentation of anaerobic microorganisms. This paper introduces the development process of SyPaB, enzymatic characteristic and steps of enzyme-catalyzed. In the end, the advantages and disadvantages of SyPaB and industrial applications are discussed.