A new carbazole derivative CNS was prepared by the condensation of 2‐hydrazine benzothiazole with 3‐aldehyde‐6‐naphthalene‐N‐butyl carbazole, which could highly selectively discriminate Co2+ and Cu2+ in different solvent systems. CNS could enable the recognition of Co2+ by the “naked eye,” with the color changing from colorless to pale yellow in DMSO/H2O (v/v = 1:1). The addition of Cu2+ made the fluorescence emission intensity increase nearly 10 times, which was accompanied by a weak blue shift of the emission peak in CH3CN. Under the illumination of 365 nm ultraviolet lamp, the color of the CNS solution changed from light blue to dark blue after Cu2+ was added. The binding process could be reasonably speculated by spectrometric titrations, 1H NMR, mass spectrometry (MS) analysis, and density functional theory (DFT) calculation. The results of fluorescence electron microscopy showed that CNS could be used as a fluorescent sensor to detect Cu2+ in Hela cells. In addition, CNS was successfully used for the detection of Cu2+ in actual water samples.
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