In recent years, researchers focused on finding green alternative media to organic solvents for enzyme-catalyzed reactions. Thereby, ionic liquids (IL) emerged as fascinating media for biotransformation. However, one drawback to their wider development is their cost, synthesis and purification. Recently, a novel medium with similar properties to IL but with additional advantages regarding cost, environmental impact and synthesis has been created: Deep Eutectic Solvents (DESs). DESs result from the association between an ammonium or phosphonium salt with a hydrogen-bond donor. Results showed the superior performance of choline chloride pair with urea or glycerol over other types of DESs in improving the conversion and selectivity of alcoholysis reaction of aliphatic ester using Candida antarctica B lipase (iCALB). We demonstrated that some DESs can react and compete with the substrates in alcoholysis reactions leading to byproduct formation and DES destruction. Although we know that iCALB denaturates in solutions of urea, it did not denaturate quickly in DESs containing urea or glycerol and its stability is sufficient to allow the reaction for several days. Finally, we opened new perspectives on the enzymatic modification of polar subtrate with this new generation of green, cheaper and easy to handle solvent in binary mixture with water.