A novel glucose biosensor, based on the modification of well-aligned polypyrrole nanowires array (PPyNWA) with Pt nanoparticles (PtNPs) and subsequent surface adsorption of glucose oxidase (GOx), is described. The distinct differences in the electrochemical properties of PPyNWA–GOx, PPyNWA–PtNPs, and PPyNWA–PtNPs–GOx electrodes were revealed by cyclic voltammetry. In particular, the results obtained for PPyNWA–PtNPs–GOx biosensor showed evidence of direct electron transfer due mainly to modification with PtNPs. Optimum fabrication of the PPyNWA–PtNPs–GOx biosensor for both potentiometric and amperometric detection of glucose were achieved with 0.2M pyrrole, applied current density of 0.1mAcm−2, polymerization time of 600s, cyclic deposition of PtNPs from −200mV to 200mV, scan rate of 50mVs−1, and 20 cycles. A sensitivity of 40.5mV/decade and a linear range of 10μM to 1000μM (R2=0.9936) were achieved for potentiometric detection, while for amperometric detection a sensitivity of 34.7μAcm−2mM−1 at an applied potential of 700mV and a linear range of 0.1–9mM (R2=0.9977) were achieved. In terms of achievable detection limit, potentiometric detection achieved 5.6μM of glucose, while amperometric detection achieved 27.7μM.