Abstract A sensor based on bovine serum albumin (BSA) and poly-o-phenylenediamine (PoPD) film modified electrode has been developed to study the interaction of BSA with nicotine. Because of the electro-synthesized convenient and the tunable conductivity of polymer, PoPD was used as the electrochemical probe. The interaction of nicotine with BSA was studied by electrochemistry and spectroscopy methods. The binding constant, obtained by differential pulse voltammetry (DPV), was consistent with the fluorescence analysis. The calibration graph for the determination of nicotine was obtained by the decrease of DPV peak current of PoPD in the presence of BSA. This constructed biosensor exhibited two linear relationships with nicotine concentration range of 1.83×10−10 to 1.01×10−3M and a detection limit of 55pM.