The novel pyrene-appended Schiff base probe L with aggregation-induced emissions (AIE) relevant to an increase in water fractions (0–90%) is synthesized and applied in sequentially detecting Cu
2+ and CN
−. The pyrene-based probe L firstly induces the excimer formation in the presence of Cu
2+. However, the process can be reversed by sequentially adding CN
−, which is demonstrated using the fluorescence “Off-On-Off” response in semi-aqueous media ethanol water (
v/
v = 7/3) under physiological pH (5 mM HEPES, pH 7.0). The Job’s plot, mass analysis,
1H NMR titrations, and density functional theory (DFT) interrogations confirm the 2:1 stoichiometry of excimer complex L–Cu
2+-L*, preferential binding atoms, and CN
− tuned complex reversibility. Based on the photoluminescence (PL) titration, the association constant of L to Cu
2+ is determined as 4.95 × 10
6 M
−1. From standard deviation and linear fittings, the detection limits (LODs) of Cu
2+ and CN
− are estimated as 219 nM and 580 nM, respectively. The practicality of Cu
2+ and CN
− detection is demonstrated using a TLC plate and a blended polymer membrane through which significant color changes under a UV lamp can be monitored. Moreover, utility of the designed probe L towards biological application with low toxicity is demonstrated by detecting Cu
2+ and CN
− inside HeLa cells. The responses of the probe to Cu(II) ions were also verified using living HeLa cells imaging.
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