<p>The compounds with an imidazole structure, DHH and DHM, were synthesized using benzoyl, ammonium acetate, 2-hydroxy-5-methylisophthalaldehyde, and 2-(3-aldehyde-4-hydroxyphenyl)- 4-methylthiazole-5-carboxylate as raw materials. The structures of probes DHH and DHM were characterized by FT-IR, ¹H NMR, ¹³C NMR, and HR-MS. This study found that DHH and DHM can rapidly and highly selectively recognize Cu²⁺ in EtOH-H₂O (1:1 v/v). The spectral characteristics of DHH and DHM were studied using fluorescence spectroscopy and UV–vis spectroscopy. The results show that the combination of DHH and DHM with Cu²⁺ produces a significant fluorescence quenching effect, and the influence of other metal ions can be neglected. Subsequently, through Job’s plot analysis, ¹H NMR titration, and DFT calculations, the binding mechanisms of probes DHH and DHM with Cu²⁺ were explored, and it was found that they coordinate in a 2:1 ratio. The experimental study on standard addition recovery showed that probes DHH and DHM could be used for the quantitative detection of Cu²⁺ in actual water samples. The probes were also successfully applied to the monitoring of Cu²⁺ in living cells.</p> Graphical abstract <p></p> <p>The compounds containing imidazole structures in probes DHH and DHM were successfully synthesized and characterized. Test strips made of DHH and DHM were used for rapid “naked-eye” detection of Cu2⁺ in an aqueous medium. The probes were successfully used to monitor Cu2⁺ in living cells and real water samples.</p>