We report a triazole appended imidazopyridine based chemosensor (probe 1) for sequential detection of Cu2+ and CN− ions using fluorometry. Probe 1 is fluorescent in aqueous acetonitrile (H2O‐CH3CN, 1:1, v/v) when common metal ions are present, but the fluorescence is quenched when Cu2+ is present. The binding mechanism, quantitative determination and the mode of interaction of Cu2+ with probe 1 are explained using fluorescence spectroscopy, electrospray ionization mass spectrometry and Density Functional Theory (DFT) calculations. The observed detection limit for Cu2+ is found to be (18.17×10−6 M) and it is comparable with those reported for other Cu2+ chemosensors. Further, the 1–Cu2+ complex formation is found to be reversible, and this property has been successfully exploited for the quantitative determination of CN− ions in aqueous acetonitrile. Addition of CN– leads to a complete recovery of fluorescence from 1–Cu2+ complex, permitting the real time detection with the lower limit of detection of CN− being 33.57×10−6 M. The observed experimental results are supported by DFT calculations. A possible application of 1–Cu2+ in fluorescence imaging of Rhizoctonia solani mycelia cells, contaminated with CN− ions, is also represented.