A novel naphthalimide-based Cu²⁺ fluorescent probe containing diglycolamine, which served as a water solubility group and provided the coordination ability to Cu²⁺, was designed and successfully synthesized. The response of the probe L to Cu²⁺ is reversible and very fast (20 s). The low detection limit of 49 nM for Cu²⁺ detection was sufficient for the detection of the maximum allowed copper levels in dietary and environmental sources. The stoichiometry of binding between L and Cu²⁺ was 1 : 1, as supported by fluorescence spectra and ESI-MS. L has been used for the imaging of Cu²⁺ in MCF-7 cells with satisfying results. The sensor L can be analyzed with a molecular logic gate. It was also found that the fluorescence of L-Cu²⁺ can be regulated by a photoacid.