A glucose biosensor was fabricated with loading of glucose oxidase (GOx) into a new organic-inorganic hybrid nanocomposite. The preparation involves formation of silica network into a Nafion (perfluorosulfonate ionomer) and subsequent loading of polyaniline grafted multiwalled carbon nanotubes (MWNT-g-PANI) onto Nafion-silica nanocomposite. Field emission scanning electron microscopy (FE-SEM) of Nafion-silica/MWNT-g-PANI composite reveals the presence of spherical silica particles (sizes in the range 250 nm-1 microm) and tubular MWNT-g-PANI particles. Chronoamperometry and cyclic voltammetry were used to evaluate the performance of biosensor towards glucose. The Nafion-silica/MWCNT-g-PANI/GOx biosensor exhibited a linear response to glucose in the concentration range of 1-10 mm with a correlation coefficient of 0.9972, good sensitivity (5.01 microA/mm), a low response time (approximately 6s), repeatability (R.S.D value of 2.2%) and along-term stability. The presence of silica network within Nafion and MWNT-g-PANI synergistically contributes to the performance of the biosensor towards the electrochemical detection of glucose.