An amperometric glucose biosensor was fabricated using electospun polyaniline fibers. Polyaniline was reacted with camphorsulfonic acid to produce a salt, which was then dissolved in chloroform containing polystyrene. Using this solution, fibers were formed and collected by electrospinning. Glucose oxidase was immobilized onto these fibers using an electrostatic layer-by-layer adsorption technique. In this method, poly(diallyldimethylammonium chloride) was used as the counter ion source. The level of adsorption was examined and evidence of layer-by-layer adsorption was obtained using a quartz crystal microbalance technique. A biosensor was fabricated from these fibers as a working electrode, and used to measure the glucose concentration accurately.