Layer-by-layer (LbL) film based glucose biosensor was fabricated with alternative layers of a nanocomposite (comprising of multiwalled carbon nanotubes (MWNTs), Au nanoparticles (Au NPs) and thiol functionalized polyaniline (PANI(SH)) and glucose oxidase (GOx). The successful formation of multilayers was confirmed by UV-visible spectroscopy. The components in the nanocomposite provide adequate electron transfer path between GOx and the electrode. A high value for the rate constant of electron transfer process (27.84 s(-1)) was observed at [GOx/Au-(SH)PANI-g-MWNT](n)/ITO electrode. The [GOx/Au-(SH)PANI-g-MWNT](n) biosensor exhibited high sensitivity (3.97 microA/mM) for the detection of glucose over a concentration range of 1-9 mM with a low detection limit of 0.06 microM.