Polyelectrolyte microspheres obtained using the layer by layer deposition method were evaluated as a novel template for enzyme immobilization and biosensor design. Polystyrene sulfonate spheres were coated with alternating layers of polyacrylic acid (PAA) and polyallylamine hydrochloride (PAH) in one system, and PAA - polyethyleneimine (PEI) in another system. Zeta potential and FTIR were used to monitor the deposition of each layer. The spheres were tested as a suitable platform for biosensor fabrication using two different model enzyme systems. The enzymes acetylcholinesterase (AChE), and horseradish peroxidase (HRP) were separately immobilized onto the surface of the polymer spheres by electrostatic forces. Inhibition tests using the pesticide paraoxon were carried out on the AChE based biosensors. Both PE systems exhibited a paraoxon detection limit of 4.6 x 10(-8) M. The PAH-PAH system retained 50% of original activity after 12 weeks, while the PEI-PAA system showed 40% stability after 9 weeks. The PAH-PAA electrodes coated with immobilized HRP exhibited a hydrogen peroxide detection limit of 3.5 x 10(-6) M with 87% retention after 10 weeks, compared to 9 x 10(-7) M for PEI-PAA spheres with 89% retention after 3 weeks. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.033208jes] All rights reserved.