A novel pyranose oxidase (PyOx) biosensor based on gold nanoparticles (AuNPs)-polyaniline(PANI)/AgCl/gelatin nanocomposite has been developed for the glucose detection. PyOx was immobilized on the surface of glassy carbon electrode (GCE) via the nanocomposite matrix. The electrode surface was imaged by scanning electron microscopy (SEM). Amperometric detection of the consumed oxygen during the enzymatic reaction was monitored at -0.7 V. After optimization studies, analytical characterization of the biosensor was carried out. The linear response of the AuNPs-AgCl/PANI/gelatin modified PyOx biosensor is found to be from 0.05 to 0.75 mM glucose with the equation of y = 2.043x + 0.253; R-2 = 0.993. Finally, proposed biosensor was used to analyze glucose content in real samples. Obtained data from the biosensing system was compared with a commercial enzyme assay kit based on spectro photometric Trinder reaction as a reference method. (C) 2009 Elsevier Ltd. All rights reserved.