The rising demand for eco-friendly and sustainable technologies in product formulation and industrial processes has led to increased interest of biotransformation processes. Conventionally, organic solvents are used in biocatalysis reactions, however, they have several drawbacks such as high volatility, inflammable and inhibition of biocatalyst activity. Ionic liquids (ILs) have also been employed in biocatalysis and demonstrate good performance, but their environmental impact remains a concern. As an alternative, deep eutectic solvents (DESs)—mixtures of a quaternary ammonium salt with a metal salt or hydrogen bond donors (HBDs)—offer advantages such as low toxicity, non-flammability, low vapor pressure, biocompatibility, biodegradability, and tunability via the alteration of salt and HBD combinations. Numerous studies have shown that the use of DESs in biotransformation represents a promising approach for enhancing reaction efficiency, improving enzyme stability, and supporting sustainable industrial practices. Thus, this chapter discusses the roles of DESs in biocatalysis, how the physicochemical properties of DESs affect biotransformation, and a comparison with conventional solvents.