Objective: A high-throughput detection method for Cu²⁺ was developed with a well designed water-soluble fluorescent probe(HWEHH) and the microplate reader. Methods: The specificity of the fluorescent probe for Cu²⁺ determination was studied with the selectivity experiment. The linear range and detection limit of the probe for Cu²⁺ determination was detected with titration experiment, and the accuracy of the probe for the detection of Cu²⁺ in water was studied with sample spiked recovery method. Results: The fluorescent probe was able to specifically detect Cu²⁺. When the concentration of peptide probe was 2 μmol/L, the linear range of Cu²⁺ detection was 0~1.5 μmol/L. The linear equation was Y=1 135.085 7X+2 048.187 6, and the detection limit was 0.036 μmol/L. The binding constant of HWEHH-Cu²⁺ complex was 1.3×106 mol/L. The linear range of high-throughput detection method was 0~1.5 μmol/L, and the recovery rate was 93.6%~101.9%. Conclusion: HWEHH peptide fluorescent probe can be used for high-throughput detection of Cu²⁺ in aqueous solution.