Abstract In the present work, a novel biosensor (GCE/RGO/PPy/NR) based on the nanocomposite of reduced graphene oxide (RGO), polypyrrole (PPy) immobilized by nitrate reductase (NR) was developed on a glassy carbon electrode (GCE). The conductive nanocomposite (RGO/PPy) was synthesized by in situ oxidative polymerization of pyrrole in the presence of RGO in acidic medium. A facile and green path was employed to synthesize RGO from graphene oxide (GO). This was performed by a novel route using Abelmoschus esculentus vegetable extract as a stabilizing and reducing agent for GO. The composite of reduced graphene oxide and polypyrrole (RGO/PPy) was deposited onto GCE with subsequent deposition of NR enzyme on the GCE/RGO/PPy to develop GCE/RGO/PPy/NR biosensor. The surface morphology and structural features of the composites were studied by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The electrochemical behavior and electrocatalytic activity of the biosensor were examined by cyclic voltammetry at different scan rates (20–100 mV s−1) in the synthetic nitrate solution. The developed bio-anode achieved a maximum current density of 4.24 mA cm−2 at a scan rate of 100 mV s−1 for 10 mM sodium nitrate solution.