Gold nanoparticle-polypyrrole nanocomposite film was electrochemically deposited in a single-step polymerization of pyrrole in the presence of 3-mercaptopropionic acid (MPA)-capped gold nanoparticles (GNPs) and p-toluenesulfonic acid (pTSA) on the surface of an indium tin oxide (ITO)-coated glass plate. The carboxyl functional groups surrounding the GNPs within the polymer matrix were utilized for the immobilization of urease enzyme through carbodiimide coupling reaction for the construction of a Urs/GNP(MPA)-PPy/ITO-glass bioelectrode for urea detection in Tris-HCl buffer. The resulting bioelectrode film was characterized by atomic force microscopy (AFM), high-resolution transmission electron microscopy (HRTEM), contact angle measurement, Fourier transform infrared spectroscopy (FTIR), and electrochemical techniques. The potentiometric response of the bioelectrode made of polymer nanocomposite films of two different thicknesses prepared at 100 and 250 mC cm(-2) charge densities, respectively, was studied towards the urea concentration in Tris-HCl buffer (pH 7.4). The thin polymer nanocomposite film-based bioelectrode prepared at 100 mC cm(-2) charge density exhibited a comparatively good potentiometric response than a thick 250 mC cm(-2) charge density film with a linear range of urea detection from 0.01 to 10 mM with a sensitivity of 29.7 mV per decade.