A new chemiluminescence system of Luminol-EDTA-CuFe₂O₄ NPs was firstly developed based on the fact that CuFe₂O₄ nanoparticals (NPs) could significantly enhance the chemiluminescence (CL) intensity of the Luminol-EDTA system. The UV-Vis spectra and the chemiluminescence spectra suggested that no new luminophor formed after the introducing of CuFe₂O₄ NPs in the Luminol-EDTA system. The possible mechanism of CuFe₂O₄ NPs participated in Luminol-EDTA system was proposed by combining with the property of CuFe₂O₄ NPs. Additionally, it was found that rutin could inhibit the chemiluminescence of the proposed Luminol-EDTA-CuFe₂O₄ NPs system. Combined with flow injection, the system was applied to the determination of rutin in rutin tablets. Under the optimal conditions, the relationship between the relative CL intensity and the logarithm of concentration of rutin was linear when the concentration of ratin ranged from 2×10^-8 mol/L to 2×10^-5 mol/L with the detection limit of 1.21×10^-9 mol/L. The recommended method has been successfully applied to the determination of rutin tablets and the recovery was between 97%-102% and RSD (c=1×10^-7mol/L, n=11) was 2.54%.