The effects of six synthetic imidazolium-based ionic liquids (ILs) on the Cu(II)-catalyzed chemiluminescence of lucigenin (Luc-CL) in the pH range 6.0-11 were investigated. Preliminary experiments found that the CL emission was strongly enhanced or inhibited in the presence of the ILs. The degree of enhancement or inhibition of the CL intensity in the presence of each IL was related to the molecular structure of the IL, the medium used, and the pH. The maximum enhancement of the CL intensity was observed at pH 9.0 (amplification factor = 443). This decrease in the pH at which maximum CL enhancement occurred and the substantial signal amplification of the Luc-CL may be related to a strong interaction between Cu(II) and the imidazolium ring of superior ILs at this pH. Additionally, the formation of IL microdomains in semi-aqueous media permitted more solubility of the product yielded by the Luc-CL reaction (N-methylacridone), which could increase the CL intensity. To obtain consistent data on the catalytic efficiency of Cu(II) in the presence of various ILs as well as the corresponding CL emission intensities, fluorescence quantum yields (Φ F) of lucigenin were measured under the same conditions. Comparison of the data pointed to the mechanism that controls the properties of Luc-CL in the presence of the Cu(II)/IL complexes. Based on the catalytic effect of the Cu(II)/IL complex and the measurement of the enzymatically generated H2O2, a novel, simple, and sensitive CL method for determining glucose with a detection limit (LoD) of 6.5 μM was developed. Moreover, this method was satisfactorily applied to the determination of glucose in human serum and urine samples. Graphical Abstract The lucigenin chemiluminescence assay for H2O2 and glucose using imidazolium-based ionic liquid derivatives/Cu(II) complexes as efficient catalysts at pH 9.0.