Abstract Fluorescent probes for detection of CN− still have many limitations, such as small Stokes shift, irreversible, and background interference, which hamper their applications for on‐site detection and bioimaging of CN−. In this work, we design a new CN−‐activatable fluorescent probe (named AHMM) containing an ESIPT (excited‐state intramolecular proton transfer) and hydrogen bond features, which show a large Stokes shift (225 nm) and molecular structural reversible detection. The probe AHMM exhibits an excellent selectivity for CN− without any interference from other anions in aqueous DMSO system. Furthermore, the mechanism of the interaction of AHMM with CN− is concluded by various experiments. The limit of detection of AHMM for CN− is calculated as low as 4.47 × 10−8 M, lower than the concentration of CN− deemed acceptable by WHO (World Health Organization). AHMM can recognize CN− in tap water quantitatively and on‐site by a smartphone APP. Moreover, food samples such as almond and cassava including CN− are visualized by fluorescence imaging. In addition, the probe shows practical applications of CN− imaging in cells and mice. This concept can be applied for designing multifunctional fluorescent probes with ESIPT and reversible characteristics for detection of CN−.
