Proteinase K (PK) is a proteolytic enzyme that has been widely used in nucleic acid purification, leather production, environmental protection, and other industrial applications. However, this biocatalyst cannot tolerate high temperatures which has severely restricted its wider application. As reported in previous studies, cholinium-based ionic liquids (ILs) have gained tremendous attention serving as a promising media to stabilize and preserve proteins, DNA, and other biomolecules due to their environmentally benign nature and biocompatibility. In this work, we chose 13 different kinds of cholinium-based ILs to examine their effects on the thermal stability and enzymatic activity of PK. We found that biocompatible cholinium-based ions with appropriately chosen anions can greatly improve the thermal stability of PK, whose melting temperature (T_m) is increased from ∼74.4 °C to 87.7 °C. However, the enzymatic activity is slightly reduced in the presence of ILs. Further comparison of our results with other literature findings suggests that kosmotropic anions of cholinium-based ILs are crucial to maintain the thermal stability of proteins. However, to achieve the best performance, the choice of IL anions is protein specific.