A novel polypyrrole (PPy)-based bilayer amperometric glucose biosensor integrated with a permselective layer has been developed for detection of glucose in the presence of interferences. It comprises of a PPy-GOx film grown, in the absence of electrolyte, as an inner layer, and a permselective PPy-Cl film as an outer layer. The PPy-GOx/PPy-Cl bilayer biosensor was effective in rejecting 98% of ascorbic acid and 100% of glycine, glutamic acid and uric acid. With an outer layer thickness of 6.6 nm, the bilayer biosensor gave nearly identical glucose response to that of a single layer PPy-GOx biosensor. The biosensor also exhibited good reproducibility (1.9% rsd, n = 10), high stability (more than 2 months), wide linear range (0.5–24 mM), low Km (8.4 mM), high Imax (77.2 μA cm−2), low detection limit (26.9 μM) and good sensitivity (3.5 μA cm−2 mM−1). The bilayer biosensor was successfully employed for glucose determination in various fruit juices.