Despite increasing interest in hydrogen (H2) as an alternative energy carrier, the current production of H2 still depends on fossil fuels. Biotechnological hydrogen production can provide a more sustainable way to generate H2. Hydrogenases are key enzymes involved in hydrogen metabolism of microorganisms with roles of H2 oxidation or evolution. They have potential applications in H2 production in vivo, in vitro and fuel cell. Important achievements have been made over the past decade in our understanding of hydrogenase and its biotechnological application as catalyst for H2 production and fuel cell. This review summarizes recent progress in the study of hydrogenases, involving strategies for biosynthesis, maturation process, isolation of novel hydrogenases, heterologous expression system, structural feature of oxygen (O2)-tolerant hydrogenases, and biotechnological applications for viable H2 technology.