A novel fluorescent probe CN3, containing 1,8‐naphthalimide and picolinate units, was synthesized, and its structure was characterized by 1H nuclear magnetic resonance spectroscopy (H NMR), 13C nuclear magnetic resonance spectroscopy (C NMR), and mass spectroscopy techniques. The detection property of CN3 toward copper ions (Cu2+) has been investigated in ethanol–HEPES buffer (v/v = 1/1, pH = 7.40) solution by UV–Vis absorption and fluorescence emission spectra. The results showed that CN3 had a highly selective and sensitive fluorescence quenching response to Cu2+, which was attributed to the generation of weak fluorescent N‐ethyl‐4‐hydroxyphenyl‐1,8‐ naphthalimide (compound 2) in polar ethanol–HEPES buffer (v/v = 1/1, pH = 7.40) via selective hydrolysis reaction. The detection of CN3 for Cu2+ was not influenced in the presence of other competing metal ions, and the limit of detection was as low as 50.0 nM. Therefore, the color of CN3 changed from colorless to yellowish when the Cu2+ was added. Furthermore, the fluorescent probe CN3 was utilized to detect Cu2+ in real water samples with fine performance.