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The conversion of biomass into biofuels has drawn considerable attention as a promising alternative for fossil fuels. In general sense, fermentation refers to the biochemical transformation from organic substances to simpler compounds by utilizing the activities of enzymes in anaerobic environment. While in industry, fermentation is a prevalent technology which converts simple sugars to alcohols (ethanol, butanol and also propanols, etc.) and other value-added products through the metabolisms of microorganisms such as yeasts and certain species of bacteria. As an important stage in biomass conversion, biofuel fermentation has been advanced significantly towards the technologies that enable commercialization. This chapter provides a comprehensive overview of the current status and prospects of biofuel fermentation (mainly bioethanol and biobutanol), including the basic concepts, microorganisms and …