A method is described for construction of a highly sensitive electrochemical biosensor for detection of glutamate. The biosensor is based on covalent immobilization of glutamate oxidase (GluOx) onto polypyrrole nanoparticles and polyaniline composite film (PPyNPs/PANI) electrodeposited onto Au electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3 s at pH 7.5 (0.1 M sodium phosphate) and 35 degrees C, when operated at 50 mV s(-1). It exhibited excellent sensitivity (detection limit as 0.1 nM), fast response time and wider linear range (from 0.02 to 400 mu M). Analytical recovery of added glutamate (5 mM and 10 mM) was 95.56 and 97%, while within batch and between batch coefficients of variation were 3.2% and 3.35% respectively. The enzyme electrode was used 100 times over a period of 60 days, when stored at 4 C. The biosensor measured glutamate level in food stuff, which correlated well with a standard colorimetric method (r = 0.99). (c) 2014 Elsevier Inc. All rights reserved.