The sensitive determination of multiple heavy metal ions and toxic anions is important in biological and environmental fields. Here we report a facile strategy to construct a multifunctional chemosensor for the detection of Hg2+,
NO
2
−
Co2+, and CN− in aqueous solution based on the fluorescent copper nanoclusters (Cu NCs). It was interesting to find that salicylaldehyde (SA) could effectively modulate the fluorescence property and sensing behavior of Cu NCs. In the absence of SA, Cu NCs showed ‘on-off’ fluorescence responses at the addition of Hg2+ and
NO
2
−
under different quenching mechanisms. Upon the presence of SA, Cu NCs exhibited a strong intramolecular charge transfer emission at 500 nm, accompanied by the decrease of the initial fluorescence of Cu NCs at 430 nm. This fluorescence on-state of Cu NC–SA at 500 nm was found to be exclusively turned off by Co2+ and enhanced by CN−. Spectroscopy results combined with thermodynamic analysis provided sufficient information to deduce the sensing mechanisms. Finally, the Cu NCs showed high biocompatibility and were able to be used for fluorescence bioimaging in living cells. This study provided a novel and simple strategy to construct the multifunctional chemosensors for bioanalytical applications.