In this study, voltammetric and spectroscopic investigation of the interaction between Janus Green B (JGB) and bovine serum albumin (BSA) was reported. The interaction was observed at Britton-Robinson buffer (pH 7.0). When JGB was added to solution containing BSA, the peak currents of BSA decrease with the increasing of JGB concentrations which is due to the interaction of JGB and BSA. The binding constant of JGB with BSA was obtained by voltammetric data. Also, this interaction was supported by means of UV-vis spectroscopic measurements. The UV-vis absorption spectra of JGB in the presence of BSA decrease with the increasing of BSA concentrations.