The development of electrochemical biosensors for controlling glucose concentration is crucial for both clinical diagnosis and the food industry. One potential technology uses reduced rGO and conducting polymers. Researchers applied a novel thermal reduction protocol to synthesize rGO from GO in the presence of malonic acid. The synthesized rGO was characterized and used in combination with polyaniline (PANI), Nafion, and glucose oxidase (GOx) to develop an amperometric glucose biosensor. The working electrode of the biosensor was a graphite rod (GR) electrode premodified with a dispersion of PANI nanostructures and rGO, Nafion, and GOx. An optimal ratio of 1:10 of PANI and rGO in the dispersion was determined for GOx immobilization. The developed glucose biosensor showed a wide linear range (from 0.5 to 50 mM), low limit of detection (0.089 mM), good selectivity, reproducibility, and stability, making it suitable for glucose determination in human serum. The PANI nanostructure and rGO dispersion showed promise for constructing electrochemical glucose biosensors. The rGO presence in the matrix improved the electrochemical performance of the glucose biosensor, making it suitable for other biosensors based on various enzymes.