beta-Glucosidase (BG) hydrolyzes cellobiose into glucose, and is a vital step in converting ionic liquids (ILs)-pretreated biomass to sustainable biofuels. The inactivation mechanism of BG from Paenibacillus sp. LLZ1 induced by microcrystalline cellulose was explored in various concentrations of ILs, composed of [Emim](+) cation and [DEP](-), [OAc](-), [Br](-), [Cl](-), and [BF4](-) anions. The FTIR analysis of inactivated BG indicated that the ILs altered its beta-sheet content. Moreover, circular dichroism spectroscopy (CD) suggested that the alpha-helix content decreased, while the beta-sheet content increased with the presence of ILs in general. Interestingly, the secondary structure of BG had almost no change after [Emim]DEP treatment, while ionic liquid [Emim]BF4 treatment caused the irreversible denaturation of BG. Eventually, by adding 0.4 mM of Aerosol OT surfactant, the BG activity was increased by 20.1% in the presence of 25% [Emim]DEP, and the corresponding glucose yield from hydrolysis of cellobiose was increased by 23.9%.