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A fluorescence "turn-on" probe for Cu (Ⅱ) ions was prepared based on the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. A strong fluorescent flavonoid intermediate was formed and verified by the NMR and ESI-MS experiments. The water-soluble and pH dependence experiments were performed to confirm the optimal solvent condition (CH 3 CN: HEPES = 1:1, v/v, pH = 7.2–7.4). The dynamic experiments indicated that the formation process of the intermediate catalyzed by Cu(Ⅱ) ions was probably pseudo-first-order reaction process. The probe showed good selectivity toward copper ions and almost no interference except Ag+ ions by the selectivity and competitive experiments. The HeLa cells were used in the cell fluorescence imaging tests and it was demonstrated that the probe could be used in the phycological condition and showed weak cytotoxicity by the MTT experiments. [Display omitted] • A Cu2+ ions biosensor was constructed by the condensation reaction of coumaraldehyde and 1-hydroxy-2-acetylnaphthalene. • The probe exhibited high selectivity for Cu2+ and could not be disturbed even if high concentration of GSH existed. • The mechanism probably involved the procedure of copper-mediated cyclization to generate the flavonoid intermediate. • The probe can be used to detect Cu2+ ions in physiological environment proven by the cell image experiments. [ABSTRACT FROM AUTHOR]