It is noteworthy that the formation of the DNA G-quadruplex is induced by factors other than stabilizing cations because this event probably occurs in living cells. Previous studies have shown that thrombin-binding DNA aptamer (TBA) forms a chair-type intramolecular G-quadruplex structure that binds with thrombin protein in the absence of stabilizing cations. Here, we used circular dichroism (CD) spectroscopy to confirm G-quadruplex formation in the presence of thrombin without stabilizing cations. We obtained characteristic CD spectra that demonstrated that TBA forms the distinctive G-quadruplex structure. Additionally, we investigated G-quadruplex formation induced by change of solvent environment: the influence of low-temperature conditions and molecular crowding.