Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, sarcosine oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosangraft-polyaniline (Fe3O4-NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe3O4-NPs. A creatinine biosensor was fabricated using Enzymes/Fe3O4-NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM). Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2s at pH 7.5 and 30 degrees C, when polarized at 0.4V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from I to 800 mu M. The sensitivity of the biosensor was 3.9 mu A mu M-1 cm(-2), with a detection limit of 1 mu M (S/N = 3). Apparent Michaelis-Menton (K-m) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 degrees C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method (r=0.99). (C) 2012 Elsevier Inc. All rights reserved.