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A novel chemiluminescence (CL) reaction was based on the oxidizing reaction of luminol by the trivalent copper-periodate complex (K5[Cu(HIO6)2], DPC) in alkaline medium. The CL intensity could be enhanced in the presence of amikacin sulfate (AKS). A new CL method was developed for the determination of AKS by coupling with flow injection (FI) technology. Because of the distinctive oxidative effect of DPC, the luminol-based CL reaction could occur at a low concentration of 10à7 M. The relative CL intensity was proportional to the concentration of AKS in the range of 4.0 ? 10à9-4.0 ? 10à6 g/mL with the detection limit of 1.2 ? 10à9 g/mL. The relative standard deviation was 2.1% for 8.0 ? 10à9 g/mL AKS (n?9). The proposed method was successfully applied to the direct determination of AKS at the level of ng/mL in serum samples. The recovery varied from 97.0% to 106.3%. A possible mechanism of the CL reaction was discussed in detail by relating to the CL kinetic characteristics and electrochemical activities of the oxidant DPC.