The selective detection of Cu2+ and S2− in pure water systems is very challenging. To address this problem, we here present a novel fluorescent probe CF536 for the reversible detection of Cu2+ and S2− in a 100% aqueous solution via a displacement approach based on fluorescence quenching and the ESIPT mechanism, and characterized by IR, NMR, HRMS and X-ray analysis. The introduction of Cu2+ (1.0 equiv.) to the aqueous solution of CF536 led to quenching of the intense green fluorescence within 2min. On the addition of S2− (3.0 equiv.), the emission of the ESIPT probe soon recovered from the CF536-Cu2+ system due to the abolishment of paramagnetic Cu2+. The fluorescence “ON-OFF-ON” cycle can be repeated a minimum of 5 times by the alternate addition of Cu2+ and S2−, implying that CF536 is a renewable dual-functional probe. The probe displayed high selectivity, a rapid response time, excellent water solubility due to introduction of the extremely hydrophilic trihydroxyl group and outstanding anti-interference ability for its large Stokes shift of 101nm (λabs=435nm, λem=536nm), thus making the method highly suitable for bioimaging. The probe CF536 was successfully used for fluorescent imaging of copper and hydrogen sulfide in living cells.