Chemiluminescence (CL)–based analysis with the advantages of simplicity,low cost,high sensitivity and selectivity has become a quite useful detecting tool in flow injection (FI),liquid chromatographic,capillary electrophoresisand microfluidic systems,making an interesting field of research for scientists in a wide variety of disciplines [1].It is well know that FI–CL analysis with luminol as luminescence probe has been widely used to study the interaction of protein with small molecules [2].In the quest for a better understanding of the binding mechanisms of metal ions to protein,the interaction of lysozyme with Cu(Ⅱ) was first described by FI–CL analysis in this work.It was found that the CL intensity of luminol–lysozyme reaction could be quenched by Cu(Ⅱ),and the decrement of CL intensity was proportional to the logarithm of Cu(Ⅱ) concentration ranging from 5 to 700 pg mL-1 with the linear equation of ΔI = 24.08lgCCu(Ⅱ) – 13.24 (R = 0.9957) and the detection limit of 1.7 pg mL-1 (3σ).The binding constant K 2.06×105 L mol-1 and the binding sites n 0.67 of Cu(Ⅱ) to lysozyme were obtained by the homemade FI–CL model [2].Asp in lysozyme has been demonstrated to bear the highest affinity for Cu(Ⅱ) [3],hence it was hypothesized that the binding of Cu(Ⅱ) to lysozyme at the site of Asp52 led to a conformational change of lysozyme,resulting in the quenching of the CL signal from luminol–lysozyme reaction.At a flow rate of 2.0 mL min-1,the whole process including sampling and washing could be completed in 36 s with the relative standard deviations of 3.5,2.6 and 2.2% for 100,300 and 500 pg mL-1 Cu(Ⅱ) (n = 7),respectively.The proposed CL method could be applied to the determination of Cu(Ⅱ) in spiked human urine,with recoveries ranging from 93.4% to 106.0%.