The present study reports the effect of an ionic liquid (IL), 1-ethyl-3-methylimidazolium ethylsulfate ([Emim][EtSO4]), on the Cu2+-catalyzed lucigenin/H2O2 chemiluminescence system in the range of pH 6.5‒10. At pH 8.0, the greatest enhancement on light emission is observed, related to a strong interaction between Cu2+ and the imidazolium ring at this pH. Furthermore, the formation of IL microdomains in the aqueous solution enhances in solubility of N-methylacridone, an insoluble emitter that can be very effective in lucigenin chemiluminescence (Luci-CL). The mentioned findings encouraged us to suggest a novel and simple method for the determination of glucose with the detection limit of 7 μM at pH 8.0 based on the detection of H2O2 liberated in enzymatic reaction between glucose oxidase and glucose by the [Emim][EtSO4]/Cu2+-catalyzed Luci-CL system. Also, the developed method was successfully applied to the determination of glucose in real plasma and urine samples of diabetic patients and validated against colorimetric spectroscopy method.