An amperometric glucose sensor based on a nanoparticulate form of polyaniline (PAni) is reported herein. The PAni nanoparticles were synthesized using pectin (PAni–Pec NPs) and functionalized with glucose oxidase (GOx-PAni–Pec NPs) to create a highly specific biosensor. Biopolymer pectin stabilizes the colloidal nanoparticles of PAni while also allowing homogenous and a high degree of functionalization of the nanoparticles with glucose oxidase via covalent coupling. This helped to arrest enzyme leaching and enhance biosensor stability. The biosensor showed high sensitivity (79.49 μA mM^-1 cm^-2), wide linear range (0.06–4 mM) and a low detection limit (43.5 μM) at a working potential of 0.6 V. The sensitivity exhibited by GOx-PAni–Pec NPs reported herein was three times higher than that of conventional polyaniline for the same amount of GOx immobilized by a physical adsorption method. Efficient loading and organization of GOx on the nanoparticles could afford adequate availability of the substrate to the enzyme sites leading to better sensitivity. The biosensor did not respond to the presence of electroactive interferents (ascorbic acid, urea, and uric acid) coexisting with glucose in physiological fluids, thereby revealing its specificity. The analytical performance of the biosensor was evaluated for estimation of glucose in blood serum samples. The reliability and stability of the glucose biosensor indicate its potential for application in routine analysis of glucose in physiological fluids.