Chemosensors play important roles in cation and anion recognition in biological, industrial, and environmental processes. Although many efforts have been made to develop artificial fluorescent receptors for Cu2+ and S2-, their applications in the detection in bulk solutions are limited. In this work, we report a novel fluorescence chemosensor (NL) based on the 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore for the quantification of Cu2+ and S2- in single intact cells. NL specifically binds to Cu2+ in the presence of other competing cations, and evident changes in UV-vis and fluorescence spectra in HEPES buffer are noticed. Based on the displacement approach, the selective sense S2- with the in situ generated NL-Cu2+ ensemble gives a remarkable recovery of fluorescence and UV-vis absorption spectra. The detection limits of NL for Cu2+ and NL-Cu2+ for S2- were estimated to be 1.6 nM and 0.17 μM, respectively. NL and the resultant complex NL-Cu2+ exhibit low cytotoxicity and cell-membrane permeability, which makes them capable of Cu2+ and S2- imaging and quantification in living MDA-MB-231 cells.