In recent years, the potential of α-chymotrypsin (CT) as biocatalysts has expanded new areas of its application ranging from pharmaceutical to chemical industries. However, attaining high thermal stability is one of the major challenges to the use of this enzyme in biocatalysis. In this regard, ionic liquids (ILs) have been used as promising media for the stabilization and preservation of proteins, enzymes, DNA and other biomolecules. In the present study, it was found that a series of cholinium-based ILs such as choline acetate ([Ch][Ac]), choline chloride ([Ch][Cl]), and choline dihydrogen phosphate ([Ch][Dhp]) stabilized the CT structure against thermal denaturation. The transition temperature (T_m) of CT was increased from ∼48.9 °C (in the buffer) to 58 °C (in the ILs media). The enzymatic activity of CT in the presence of ILs was also monitored by using casein as the substrate. It was found that choline dihydrogen citrate ([Ch][Dhc]) and choline hydroxide ([Ch][OH]) dramatically decreased the enzyme activity. Both structural stability and enzymatic activity were retained in [Ch][Ac], [Ch][Cl] and [Ch][Dhp], indicating the suitability of these ILs as a high-temperature bio-catalytic reactor systems. Our results revealed that [Ch][Ac] is the best stabilizer among all studied ILs for the native structure of CT, whereas [Ch][OH] is the strongest destabilizer for the CT structure. The outcome of our results can be helpful to overcome some of the major limitations found in the development of biocatalytic processes.