Hydrogenases are the most important enzymes in biological hydrogen production and hydrogen energy utilization. They are widely distributed, oxygen-sensitive, multiunit complexed metal enzymes. In vitro synthetic enzymatic biosystems (ivSEB) is a type of in vitro biotransformation (ivBT) technology, which is an emerging biomanufacturing powerhouse that combines microbial fermentation with enzymatic biocatalysis, allowing for novel and efficient hydrogen production, also breaking the Thauer limit and achieving a yield of hydrogen close to the theoretical value of chemistry (1 mole of glucose to produce 12 moles of hydrogen in maximum). It represents the future direction of biological hydrogen production. However, the recombinant expression of hydrogenase is the main bottleneck limiting the wide application of ivSEB for hydrogen production technology. Hydrogenases are widely distributed in all life domains, but are oxygen-sensitive and mostly consist of metalloproteins with multi-subunits, bearing [Fe] only, [NiFe] or [FeFe] dinuclear core in their catalytic center. Oxygen not only inhibits the activity of hydrogenase, but also affects the transcription of the enzyme-encoding gene and post-translational process of the enzymes. As a result, the levels of recombinant hydrogenase are usually low and the enzymatic activities are also incomparable to the native enzymes, often leading to high production costs due to the strict anaerobic purification procedures. In order to meet the requirements of industrial hydrogen production, hydrogenases must possess excellent catalytic properties, such as a high catalytic turnover number, great thermal stability, and the ability to tolerate trace amounts of oxygen. This review summarizes the studies on the structural and catalytic characterizations of hydrogenases, including their classification, oxygen resistance mechanisms, and progress in recombinant expression. Additionally, the evolution of natural electron transfer chains and the design of artificial routes, which can improve hydrogen production efficiency and reduce costs, are briefly discussed. The review also discussed the progress in the studies on the mechanisms of hydrogenases’ tolerance toward oxygen, the strategies for microbial expression of recombinant hydrogenases as well as the optimization of the artificial electron transfer chains adapted for the production of hydrogen using ivSEB, in expectations of promoting the applications of hydrogenases involved ivSEB, from renewable energy storage, anaerobic artificial respiration, to clean hydrogenation or dehydrogenation in biocatalysis.