In this paper, the interaction of rutin (Rt) with rifampicin (RIF) has been investigated using voltammetric and spectroscopic techniques. With the addition of RIF into the Rt solution, both the reduction and oxidation currents of rutin decrease with few changes in the peak potentials and no new peaks appeared. The binding of Rt with RIF forms a kind of supramolecular complex Rt-RIF, which is electrochemically non-active. The experimental data showed that the formation of supramolecular complex is due to the electrostatic attraction of Rt with RIF. The binding reaction resulted in the decrease of the concentration of free Rt in the solution, and the decrease of the cathodic peak current of Rt. The stoichiometry of the Rt-RIF biocomplex was determinated to be 1: 1 by means of voltammetric data. The effect of pH on the binding reaction has been also studied. Based on the peak current values of Rt in the presence and absence of RIF, the binding constants of Rt-RIF at pHs 4, 7 and 9 were calculated as 0.72 x 10(4) M-1, 1.28 x 10(4) M-1 and 0.19 x 10(4) M-1, respectively. The experimental results indicate that there is a strength interaction between Rt and RIF in neutral pH. This binding reaction was supported by UV-Vis. and IR spectroscopy techniques.