Polythymine (poly T)-templated copper nanoparticles (CuNPs) were demonstrated as novel and sensitive fluorescence probes for the detection of Pb(2+) based on the fluorescence quenching effect. The as-prepared CuNPs displayed strong fluorescence emission. However, the fluorescence of CuNPs was readily quenched in the presence of Pb(2+). These changes in fluorescence intensity of CuNPs allowed for the analysis of Pb(2+) with rapidity (<10 min), simplicity (label-free), high sensitivity (LOD 0.4 nM), high selectivity (no interference from other metal ions) and at low-cost (without any labels and sophisticated operation). We validated the practicality of using CuNPs for the determination of Pb(2+) in environmental samples through analyses of tap water samples.