The interactions between a flavonoid, rutin (Rt), and basic L-amino acids (BAAs) were studied at physiological pH (7.40) by UV-Visible spectroscopy and voltammetric techniques. The binding stoichiometries and constants (K) at different temperatures were measured by voltammetric method. So, the thermodynamic parameters Delta H (au <), Delta G (au <), Delta S (au <) were calculated and the results indicate that the binding reaction is mainly entropy-driven and hydrophobic forces played major role in the interactions of arginine and lysine with rutin as compared to that of histidine. Whereas, the histidine binding reaction, including with the negative enthalpy and entropy changes is entalpy-driven and this interaction primarily arise from hydrogen-bonding and van der Waals interactions. Finally, it was also observed that the affinity (that is binding constant) of hydrogen-bonding and van der Waals interactions between Rt and arginine or lysine decreased, but the stability of hydrophobic interaction of Rt with histidine increased when the temperature was raised.