To meet the severe water pollution status, multi-target biosensor becomes one of research focus. We herein report a novel duplex functional fluorescent biosensor for the label-free and separate detection of Pb2+ and Hg2+ with high sensitivity and selectivity. A K+-induced fluorescent G-quadruplex probe was assembled by a guanine-rich sequence (AGRO100) and N-methyl mesoporphyrin IX(NMM). It changed into a more stably non fluorescent G-quadruplex structure and a hairpin-like structure upon binding Pb2+ and Hg2+ ions, respectively. As a result, the fluorescence decreased with relation to the addition of targets, allowing the separate detection of Pb2+ and Hg2+ ions at concentrations as low as 5 nM and 18.6 nM. The linear correlation existed between the fluorescence intensity and the concentration of Pb2+ and Hg2+ over the range of 10-200 nM (R-2 = 0.98) and 20-1000 nM (R-2 = 0.99), respectively. All real lake samples, even containing Pb2+ or Hg2+ at 50 nM, could be determined by the duplex functional fluorescent probe with recovery rates 96% and 104%, respectively. Considering that this sensor is label-free, simple, time-saving, cost-effective and easy-to-handle, it is possible to pave its way for Pb2+ and Hg2+ detection in various application fields.