In this study, we developed a fluorescence assay for the highly sensitive and selective detection of Hg(2+) and Pb(2+) ions using a gold nanoparticle (Au NP)-based probe. The Hg-Au and Pb-Au alloys that formed on the Au NP surfaces allowed the Au NPs to exhibit peroxidase-mimicking catalytic activity in the H(2)O(2)-mediated oxidation of Amplex UltraRed (AUR). The fluorescence of the AUR oxidation product increased upon increasing the concentration of either Hg(2+) or Pb(2+) ions. By controlling the pH values of 5mM tris-acetate buffers at 7.0 and 9.0, this H(2)O(2)-AUR-Au NP probe detected Hg(2+) and Pb(2+) ions, respectively, both with limits of detection (signal-to-noise ratio: 3) of 4.0 nM. The fluorescence intensity of the AUR oxidation product was proportional to the concentrations of Hg(2+) and Pb(2+) ions over ranges 0.05-1 μM (R(2)=0.993) and 0.05-5 μM (R(2)=0.996), respectively. The H(2)O(2)-AUR-Au NP probe was highly selective for Hg(2+) (>100-fold) and Pb(2+) (>300-fold) ions in the presence of other tested metal ions. We validated the practicality of this simple, selective, and sensitive H(2)O(2)-AUR-Au NP probe through determination of the concentrations of Hg(2+) and Pb(2+) ions in a lake water sample and of Pb(2+) ions in a blood sample. To the best of our knowledge, this system is the first example of Au NPs being used as enzyme-mimics for the fluorescence detection of Hg(2+) and Pb(2+) ions.