tSensitive and selective detection of Pb2+is of great importance to both human health and environ-mental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb2+in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probefor signaling Pb2+. It was found that the aptamer probe had a high FA in the absence of Pb2+. This isbecause the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases,which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb2+to formG-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of thefluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly uponbinding to Pb2+. Indeed, we observed a decrease in FA of aptamer probe upon Pb2+binding. Circulardichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliabil-ity and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, wewere able to real-time detect binding between the TBA probe and Pb2+. Moreover, the aptamer probewas exploited as a recognition element for quantification of Pb2+in homogeneous solution. The changein FA showed a linear response to Pb2+from 10 nM to 2.0 M, with 1.0 nM limit of detection. In addition,this sensing system exhibited good selectivity for Pb2+over other metal ions. The method is simple, quickand inherits the advantages of aptamer and FA.