Ionic liquids (IL) are used as a new class of solvents for various reactions. Especially using IL in biocatalysis in an aqueous milieu has attracted considerable attention because enzymes show remarkable differences in their catalytic features in IL-containing reaction media. Firefly luciferase is widely used in many analytical techniques, because light production of firefly luciferase is one of the most sensitive analytical measures in the ultrasensitive detection of adenosine-5′-triphosphate, e.g. for measuring microbial contamination and monitoring gene expression, as well as for monitoring tumor growth and metastasis in whole animals. Firefly luciferase is an unstable enzyme and its inactivation can lead to low sensitivity in the above-mentioned assays. The present study addresses the comparative influence of six different water-immiscible IL, the 3-methylimidazolium derivatives [BMIM]Cl, [HMIM]Cl, [BMIM]Br, [EMIM]Br, [HMIM]Br, and [BMIM]BF4, on the kinetic properties, structural stability, and function of firefly luciferase from Photinus pyralis using circular dichroism, fluorescence spectroscopy, and a bioluminescence assay. The incubation of luciferase with various IL showed that, with the exception of [BMIM]BF4, the activity and stability of luciferase was considerably increased in the presence of IL, compared to luciferase in aqueous medium. Moreover, Km for the substrate adenosine-5′-triphosphate in the presence of IL (except for [BMIM]BF4) decreased while Km for luciferin remained constant.