Lead (Pb2+) ions give an imminent danger since they have been known to cause persistent damage to humans, plants, and animals, even at low concentrations, and cysteine (Cys) elevated levels are critical indicators for many diseases. Therefore, their detection is critical in pharmaceutical and environmental samples. This study tailored an innovative fluorescence switch off-on assay to detect Pb2+ and Cys based on the amplification of G-quadruplex (G-4) to N-methylmesoporphyrin IX (NMM). This assay operates on the fluorescence of NMM serving as a signal reporter which could be enhanced by an adenine-guanine-rich probes G-4. Initially, the fluorescence of NMM was increased after binding with G-4 and Pb2+ and effectively quenching fluorescence without altering the structure of G-4. As it was proved by Circular dichroism (CD). The number of binding sites for Pb2+ per NMM was determined to be 0.80 with a binding constant of 1.9 x 10(4) mol /L. The presence of Cys may disrupt the interaction between Pb2+ and G-4/NMM due to its stronger binding affinity towards Pb2+ leading to high fluorescence recovery.The assay demonstrated the capability to detect Pb2+ within a concentration range of 0.4 to 1.6 mu M, achieving a high correlation coefficient (R-2 = 0.985). with the detection limit of 0.45 mu M was established. Similarly, Cys was effectively detected across a range of 1 to 6 mu M, possessing correlation (R-2 = 0.973) with a detection limit of 1.51 mu M, further confirming that the detection limit is not influenced by the starting point of the linear range. The assay detected these compounds among various other amino acids and heavy metals. Our approach is simple and innovative, enabling the accurate determination of Pb2+ and Cys concentrations in soil and medicinal samples, highlighting its potential in practical diagnostic and environmental applications.