In this paper, an innovative and facile one-pot method for synthesizing water-soluble and stable fluorescent Cu nanoclusters (CuNCs), in which glutathione (GSH) served as protecting ligand and ascorbic acid (AA) as reducing agent was reported. The resultant CuNCs emitted blue-green fluorescence at 440 nm, with a quantum yield (QD) of about 3.08%. In addition, the prepared CuNCs exhibited excellent properties such as good water solubility, photostability and high stability toward high ionic strength. On the basis of the selective quenching of Hg on CuNCs fluorescence, which may be the result of Hg ion-induced aggregation of the CuNCs, the CuNCs was used for the selective and sensitive determination of Hg in aqueous solution. The proposed analytical strategy permitted detection of Hg in a linear range of 4 × 10 to 6 × 10 M, with a detection limit of 2.2 × 10 M. Eventually, the practicability of this sensing approach was confirmed by its successful application to assay Hg in tap water, Lotus lake water and river water samples with the quantitative spike recoveries ranging from 96.9% to 105.4%.