In this study, a very thin film of biocompatible gelatin B (GB) fabricated onto indium tin oxide (ITO)-coated glass substrate for electrochemical catalytic activity towards different metabolites has been investigated. The optical and electrochemical properties of bare GB/ITO electrode and with different metabolites were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and electrochemical techniques. The optical properties clearly indicate the structural and surface morphological changes on electrode surface. FTIR spectra showed displacement of the IR peaks towards smaller wave numbers, indicating possible existence of hydrogen bonding between the GB and metabolites. The catalytic behaviour of GB/ITO electrode towards ascorbic acid (AA), citric acid (CA), oxalic acid (OA), glucose (Glu), sucrose (Suc), lactose (Lac) and fructose (Fru) has been investigated by cyclic voltammetry (CV). The electrochemical response studies of GB/ITO electrode have been monitored with different metabolites in the range of 10-500 mg/dl. The sensitivity of GB/ITO electrode for AA and OA was found as 0.156 and 0.108 mu A/(mg/dl cm(-2)) respectively. The results indicate that the GB/ITO electrode has higher specificity towards the AA and OA. The attractive properties of GB/ITO electrode provide the potential applications in the simultaneous detection of AA and OA. The excellent electrocatalytic behaviour of GB/ITO electrode may be useful towards the construction of electrochemical biosensors.