A one-pot sonochemical reaction of Cu(NO3)2 with glutathione (GSH), the latter functioning as a reducing agent and a stabilizing agent, rapidly affords Cu nanoclusters (NCs). The as-prepared GSH-CuNCs possess a small size (∼2.2 ± 0.2 nm), red luminescence with quantum yield (5.3%), and water-dispersibility. Moreover, the fluorescence of the as-prepared GSH-CuNCs is responsive to pH so that the intensity of fluorescence increases rapidly with decreasing pH from 9 to 4. Besides, the GSH-CuNCs would be aggregated by Pb(2+) ions in aqueous solution which results in quenching of the fluorescence. Therefore, such GSH-CuNCs would be excellent candidates as fluorescent probes for the label-free detection of Pb(2+) with the limit of detection at 1.0 nM. Importantly, CAL-27 cells are used as models to achieve potential application as probes for monitoring Pb(2+) in living cells. Thus, these fluorescent CuNCs could work as an alternative to conventional fluorescent probes for biolabeling, sensing and other applications.