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In this paper, a novel fluorescence biosensing strategy based on GR-5 DNAzyme has been developed for the simple, rapid, sensitive and selective detection of lead ions (Pb2+) by using gold nanoparticles (AuNPs) as the fluorescence quencher. Data from dynamic light scattering (DLS) and zeta potential measurements demonstrated that GR-5 DNAzyme functionalized AuNPs were successfully prepared with good dispersion in aqueous solution. The carboxyfluorescein (FAM) labeled GR-5 DNAzyme was in close proximity to the AuNPs and the fluorescence signal was quenched, when the DNAzyme was modified on the surface of AuNPs. In the presence of Pb2+ ions, the GR-5 DNAzyme was activated and cleaved the substrate strand at the RNA site (rA) into two parts, which led to a short FAM-linked oligonucleotide fragment being released from the surface of the AuNPs, and the fluorescence intensity was gradually recovered. By taking advantage of the high fluorescence quenching efficiency of AuNPs, our proposed method exhibited a high sensitive detection of Pb2+ ions with a detection limit of 250 pM and a linear range from 1 nM to 100 nM. With the choice of a classic GR-5 DNAzyme instead of 8-17 DNAzyme as the catalytic unit, the selectivity of this strategy was obviously improved compared with previously reported methods. Moreover, this biosensing strategy was also used for the determination of Pb2+ in lake water samples with satisfying results. The strategy would find potential applications in environmental and biomedical fields. [ABSTRACT FROM AUTHOR]