We describe the fabrication of an amperometric biosensor based on cytochrome c (Cyt c) immobilized graphene oxide–multiwalled carbon nanotube (GO–MWCNT) composite on a nano Au modified glassy carbon electrode for trace level detection of H₂O₂. Morphology and surface characterization of the nanocomposite reveal the successful formation of a highly conducting MWCNT network on the GO surface. Electrochemical impedance studies indicate a lower charge transfer resistance compared to the bare electrode. Cyclic voltammetry studies clearly demonstrate an enhanced direct electrochemistry of Cyt c with a high electron transfer rate constant (k_s) value of 3.4 s⁻¹. An amperometric H₂O₂ biosensor has been fabricated with an excellent current sensitivity of 0.533 μA pM⁻¹ cm⁻² and a very low detection limit of 27.7 pM. The fabricated sensor shows exceptional selectivity to H₂O₂ in the presence of a high concentration of some likely interferents. Moreover, the sensor exhibits high stability with appreciable repeatability and reproducibility.