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The interaction between sanguinarine and guanosine was investigated by using UV-vis and fluorescence spectra at pH 7.2. The binding of sanguinarine to guanosine was substantiated by the hypochromism and bathochromism in the absorption spectra and the emission quenching in fluorescence spectra. The fluorescence lifetime results, the varieties of the fluorophore absorption spectra and the decrease of the binding constant with the increasing temperature all indicate that the fluorescence quenching is static. The ratio and constant of the binding cytidine to sanguinarine are 2 and 6.44 x 107, respectively. The result shows that the binding of sanguinarine to guanosine is not only exothermic but also entropy-driven with △H= -8.53 kJ/mol, AS = 0.12 kJ/(mol K), and △G =-44.57 kJ/mol at 298.15 K.