It has been shown that guanine-rich DNA can fold into a G-quadruplex with certain metal cations. The spectral characteristics, thermo-stability, and kinetics for the formation of a Pb2+-driven G-quadruplex of thrombin-binding aptamer (TBA) were measured in the current work using a combination of ultraviolet (UV) and circular dichroism (CD) spectroscopy along with stopped-flow technique. CD spectra demonstrated that TBA could fold into a unique G-quadruplex with a strong positive peak at 312 nm. Analysis of the titration data reveals that the binding stoichiometry is 1:1 for the titration of TBA with Pb(NO3)(2), which is in accordance with the localization of the Pb2+ ion between the adjacent G-quartets. Thermal denaturation profiles indicate that the Pb2+-induced intramolecular G-quadruplex is more stable than those driven by Na+ or K+ ions. Kinetic studies suggest that the Pb2+-induced folding G-quadruplex of TBA probably proceeds through the rapid formation of an intermediate Pb2+-TBA complex, which then isomerizes to the fully folded structure. Conformational changes transpire after the addition of Pb(NO3)(2) to the Na+- or K+-induced G-quadruplexes, which may be attributed to the replacement of Na+ or K+ ions by Pb2+ ions and the generation of a more compact structure of the Pb2+-TBA structure. The relaxation time, tau, of folding the G-quadruplex is reduced from 1.05 s in the presence of Pb2+ ions alone to 0.34 s under the cooperation of initially added Na+ ions, while tau is increased to 8.33 s under the competition of initially added K+ ions.