Copper pollution has become a very serious problem in modern society with the increasing industrial emission and the acid mine drainage. Copper deficiency or excess copper accumulation in human and animal livers can bring both oxidative stress and severe disorders. Thus, the development of a simple and efficient strategy for Cu²⁺ ion detection in water and blood samples is very necessary and meaningful. In this study, a simple, fast, and sensitive fluorescence method was put forward to detect Cu²⁺ ions based on C5 DNA-templated gold nanoclusters (AuNCs) and Fenton-like reactions. Under aerobic conditions, ascorbic acid is not only involved in the reduction of Cu²⁺, but also reacts with O₂ to produce H₂O₂. Then, hydroxyl radical (˙OH), yielded by H₂O₂ and Cu⁺, can seriously quench the fluorescence of AuNCs. Attributed to the unique Fenton-like reaction between ascorbic acid and the Cu²⁺ ions, high selectivity was achieved for Cu²⁺ ion monitoring using this nanosensor, and the practical applications of this nanosensor in real water and blood samples were realized.