This work aims to develop a new cheap-facile and bifunctional and multi-responsive fluorescent sensor, which based-on isophthaloyl dichloride/nitro-benzofurazan (isophthalo-NBD), for toxic anions and cations. This paper is based on fluorometric&colorimetric methods for the determination of Fe3+&Cu2+ and F−&CN− ions through an easy-to-make sensor having double binding modes in aqueous media. The binding mode of the sensor, isophthalo-NBD, with Fe3+&Cu2+ and F−&CN− ions was confirmed by classical spectroscopic data such as absorption, fluorescence intensity, Job plot, response time, the limit of detection, COSY-NMR and 1HNMR titrations, etc. The sensor showed a remarkable color change from neon yellow to bright-red along with both an increase in fluorescence intensity, and a red-shift of emission wavelength in the presence of F− and CN− anions, respectively. Otherwise, the sensor exhibited an observable quenching effect without changing wavelength in the presence of Fe3+ and Cu2+, respectively, via the Chelation Enhanced Fluorescence effect (CHEF). The Alamar Blue method was used to determine cell viability by treating HeLa and Hep-2 cells with isophthalo-NBD. The fluorescence images of isophthalo-NBD in living cells to detect the presence of F− and CN− anions and Fe3+ and Cu2+, respectively, were performed by using a confocal laser scanning microscope.