Monitoring of kanamycin residue has attracted considerable attention owing to the potential harm caused by the abuse of kanamycin. However, the detection of kanamycin has been limited owing to its electrochemical and optical inertness. Herein, we report a facile and highly efficient electrochemiluminescence (ECL) strategy for the detection of kanamycin based on the valence state effect of gold nanocluster (AuNC) probes. It is proven that Au0 in chemically reduced AuNCs (CR-AuNCs) could be oxidized to AuI via the redox reaction between kanamycin and CR-AuNCs in the presence of H2O2, resulting in ECL quenching due to the valence state change of CR-AuNCs. Because the ECL of the AuNC probes is sensitively affected by the valence state, excellent sensitivity for kanamycin was achieved without any signal amplification operation and aptamers. A preferable linear-dependent curve was acquired in the detection range from 1.0 × 10-11 to 3.3 × 10-5 M with an extremely low detection limit of 1.5 × 10-12 M. The proposed kanamycin sensing platform is very simple and shows high selectivity and an extremely broad linear range detection of kanamycin. Furthermore, the proposed sensing platform can detect kanamycin in milk samples with excellent recoveries. Therefore, this sensing strategy provides an effective and facile way to detect kanamycin and can help promote the understanding of the constructed mechanism of the AuNC-based ECL system, thus greatly broadening its potential application in ECL fields.