Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-l-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the l-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the l-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the l-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through l-sorbose pathway and D-gluconate pathway is discussed.