G-rich single-stranded DNA (5′-TTAG-GG-3′) adopted a G-quadruplex structure in buffercontaining potassium ions. The spectroscopic featureand the interaction between methylene blue andG-quadruplex have been investigated by circular di-chroism, and nuclear magnetic resonance spectros-copy. The UV-Vis absorption and fluorescence spec-tral results show that the fluorescence behavior ofMB by single-stranded DNA fits Stern-Volmer staticquenching equation very well and they formed 1︰1complexes; dimeric G-quadruplexes were bound toMB with 1︰1 or 2︰1, and their equilibrium constantswere 1.047×105 and 8.79×104 L/mol, respectively.Based on the above results and 1H-NMR spectraldata, one may conclude that MB stacked either theterminal tetrads to form 1︰1 complexes or betweentwo terminal tetrads of G-quadruplexes to form 1︰2sandwich complexes with G-qudruplexes. G-rich single-stranded DNA (5’-TTAG-GG-3 ’) adopted a G-quadruplex structure in buffer containing potassium ions. The spectroscopic feature and the interaction between methylene blue and G-quadruplex have been investigated by circular di-chroism, and nuclear magnetic resonance spectros-copy. The UV-Vis absorption and fluorescence spec-tral results show that the fluorescence behavior of MB by single-stranded DNA fits Stern-Volmer staticquenching equation very well and they formed 1: 1 complexes; dimeric G-quadruplexes were bound toMB with 1: 1 or 2: 1, and their equilibrium constantswere 1.047 × 10 5 and 8.79 × 10 4 L / mol, respectively. Based on the above results and 1H-NMR spectral data, one may conclude that MB stacked either theterminal tetrads to form 1 : 1 complexes or betweentwo terminal tetrads of G-quadruplexes to form 1: 2sandwich complexes with G-qudruplexes.