Objective: This study aimed to developed a novel fluorescent enzyme-linked immunosorbent assay method for the sensitive detection of E. coli O157:H7 in milk samples. Methods: The pyrophosphate-Cu²⁺ coordination complex was hydrolyzed by alkaline phosphatase, and Cu²⁺ was released from copper nanoclusters as signal reporting probes. Key factors such as Cu²⁺ concentration, pyrophosphate concentration, and DNA template concentration and length were optimized. Results: Under the control of the optimized conditions, the method exhibited a good linear relationship (R²=0.980 8) with the target E. coli O157:H7 in the range of 5×10⁴~1×10⁸ CFU/mL. The limit of detection was 2.4×10⁴ CFU/mL. Conclusion: The method was successfully applied to the determination of E. coli O157:H7 in samples of low-fat milk and skim milk. The average recovery was 92.2% ~ 108.5% with relative standard deviation 4.9% ~ 10.4%.