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An effective ratiometric fluorescent probe based on silicon particles/gold nanoclusters (SiNPs/AuNCs) nanohybrid has been fabricated and applied to be a "on-off-on" switch sensing platform for detection of Hg2+ and cysteine. In this elaborated sensing platform, the SiNPs just acted as internal reference signal, providing a build-in correction for background interferences and environmental effects, to which the AuNCs as a signal report unit for Hg2+ response was covalently grafted by amidation reaction. The fluorescence intensity of SiNPs/AuNCs could be effectively quenched upon adding Hg2+, accompanied with an easily distinguishable fluorescent color change. The ratiometric fluorescence signal (F 649 /F 511) of the established nanoprobe was linearly proportional to the concentration of Hg2+ ranging from 0.02 to 24 μM with a low detection limit of 5.6 nM, which is below the guideline value of Hg2+ in drinking water set by the World Health Organization. Interestingly, upon addition of cysteine, the Hg2+-quenched fluorescence intensity was recovered gradually. Furthermore, the approach developed has also been utilized for Hg2+ detection in real complex biological samples with satisfactory results. More importantly, benefiting from the good water-solubility and excellent biocompatibility, this nanoprobe can monitor the intracellular Hg2+ and cysteine in living cells, indicating its potential applications in advanced biosensing and bioimaging. Herein, we reported an effective dual-emission ratiometric probe for "on-off-on" detection of Hg2+ and cysteine. Image 1 • An efficient dual-emission fluorescent probe for simultaneously and visual detecting Hg2+ and L-Cys was constructed. • The probe could simultaneously detection of Hg2+ in human serum and urine samples. • Due to the ratiometric fluorescence model, the background interference was extincted. • The developed probe was successfully used for the fluorescence bioimaging of Hg2+ and L-Cys in live cells. [ABSTRACT FROM AUTHOR]