We report a novel, selective and sensitive strategy for the sequentially “ON–OFF–ON” fluorescent detection of Cu2+ and S2− based on a fluorescein derivative, FL. The specific binding of FL towards Cu2+ in aqueous and biological media led to the intensive green fluorescence quenching and a notable increase of the absorbance maximum at 480nm. In the presence of S2−, the intensity and overall pattern of the fluorescence emission and UV–vis spectra of FL–Cu2+ ensemble were recovered since the abolishment of paramagnetic Cu2+. This displacement approach exhibited highly specificity, and sensitivity with detection limits of 3nM for Cu2+ and 150nM for S2−. The fluorescence “ON–OFF–ON” circle can be repeated to a minimum of 5 times by the alternative addition of Cu2+ and S2−, implying that FL is a renewable dual-functional chemosensor. The biocompatibility of FL toward breast carcinoma cells, MDA-MB-231 was confirmed by MTT assay. The reversible “ON–OFF–ON” fluorescent response of FL to Cu2+ and S2− in living system was further confirmed by confocal fluorescence imaging of living cells. The quantification of Cu2+ and S2− in single intact cell was realized by the flow cytometry analysis.