The effects of ionic liquids (ILs) on the catalytic activity of enzymes were studied by approaches of electrochemistry and quantum chemistry Calculation in this work. Three types of ILs, namely, [bmpyri]BF4, [bmpyrro]BF4, and [bmim]BF4, were selected to address the effects of different types of ILs on the electrocatalytic activity of glucose oxidase (GOx) toward the oxidation of glucose. ILs and GOx were assembled on the surface of an electrode via single-walled carbon nanotubes (SWNTs) and poly(sodium 4-styrenesulfonate) (PSS) utilizing the electrostatic interaction. Spectroscopic results indicated that ILs did not affect the conformation of the enzyme. The cyclic voltammetric results showed that the electrocatalytic activity of the GOx-IL-PSS-SWNTs/GC electrode was lower than that of the GOx-SWNTs/GC electrode. The characteristic kinetic constants of the enzymatic reaction were evaluated from the cyclic voltammograms Under a substrate-saturated condition. The values of the characteristic rate constant obtained at each IL-containing enzyme electrode were lower than those obtained at the GOx-SWNTs/GC electrode and decreased following the sequence of SWNTs > [bmpyri] > [bmpyrro] > [bmim]. The theoretical calculations combined with experiments were employed to address the interaction between the IL-s and SWNTs, showing that the presence of IL. oil the surface of SWNTs could significantly affect the electrical transfer properties of the nanotube and led to the decrease of the electrocatalytic activity of the GOx-IL-PSS-SWNTs/GC electrode. These results indicate that the nature of ILs is the main factor, which affects the electrocalalytic activity of the GOx-IL-PSS-SWNTs/GC electrodes toward the oxidation Of glucose. The results of this study are directly relevant for those applications where ILs are employed in the form of thin films supported on solid surfaces, such as the designing of the related biosensor, microelectronic devices in the ILs-containing system, and so forth.