A new nucleic acid-based fluorescence sensor is reported for simultaneous detection of Pb2+, Ag+, and Hg2+ based on the specific catalytic activity of Pb2+ for a particular DNAzyme, specific regulation of Ag+ on “C-Ag+-C” complex, and stable complex formed by Hg2+ and rhodamine B isothiocyanate (RBITC). Three fluorescence dyes, aminomethylcoumarin acetic acid (AMCA), 5-carboxyfluorescein (FAM), and RBITC, were modified on the probes and served as fluorescent donors. Upon DNA interaction with these metal ions and AuNP fluorescence quenching effect on the fluorescence dyes,the fluorescencerecovery of RBITC and the fluorescencequenching of AMCA and FAM were monitored to detect Hg2+, Pb2+, and Ag+, separately, without the need of using any masking reagents. This sensor exhibited high sensitivity and selectivity. The limit of detection (LOD) is 0.48 nM for Pb2+, 0.23 nM for Ag+, and 0.17 nM for Hg2+. Finally, this sensor was successfully applied for simultaneous detection of Pb2+, Ag+, and Hg2+ in real sample.