Abstract Many Pb2+ biosensors based on Pb2+-specific RNA-cleaving DNAzyme have been developed in the past years. However, many of them have limited practical use because of high cost (e.g., enzymes), complicated processing and the use of unstable molecules (e.g., RNA). In this study, a novel label-free fluorescent biosensor for Pb2+ was proposed based on Pb2+-induced allosteric G-quadruplex (PS2.M). In the presence of K+, N-methyl mesoporphyrin IX (NMM) could bind to K+-stabilized G-quadruplexes, giving rise to high fluorescence. On addition of Pb2+, Pb2+ competitively binded to K+-stabilized G-quadruplexes to form more compact DNA folds. The Pb2+-stabilized G-quadruplexes did not bind to NMM, which resulted in fluorescence decrease. This allowed us to utilize PS2.M for quantitative analysis of Pb2+ using the NMM–G-quadruplex system by convenient “mix-and-detect” protocol. The fluorescence emission ratio (F0/F) showed a good linear response toward Pb2+ over the range from 5.0nM to 1.0μM with a limit of detection of 1.0nM. This proposed biosensor was simple and cost efficiency in design and in operation with high sensitivity and selectivity. We validated the practicality of this biosensor for the determination of Pb2+ in lake water samples.