The interaction of papain with peptide immobilized on the Au surface altered the interfacial electron transfer resistant, charge transfer resistance (RCT), by preventing the redox species approaching the electrode was characterized by electrochemical impedance spectroscopy (EIS), AC voltammetry, cyclic voltammetry and surface plasmon resonance techniques (SPR). From cyclic voltammetry a potential shift of 25–30 mV was observed after the addition of papain, with decrease in current and the impedance results showed that RCT increased with increasing concentration of papain indicating the interaction of papain with the immobilized film. This change in kinetics was also observed in SPR. The interaction was further confirmed by reflection-absorption infrared spectroscopy (RAIR).