A new biosensor based on immobilization of glucose oxidase on a multiwalled carbon nanotube-gold nanoparticle coated glassy carbon electrode (GCE) was fabricated for the electrochemical determination of glucose. First, a carboxylated multiwalled carbon nanotube (cMWCNT) was functionalized with 4-aminothiophenol (ATP) by a coupling agent, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and then gold nanoparticles (AuNPs) were covalently linked to the ATP-cMWCNT via the formation of a covalent Au-S bond. Finally, glucose oxidase was immobilized simultaneously by the electropolymerization of o-phenylenediamine on the AuNP-ATP-cMWCNT coated GCE. The biosensor showed a linear range from 0.05 mM to 8.85 mM with a sensitivity of 27.93 μA mM-1cm-2and a detection limit of 0.015 mM for the detection of glucose. The biosensor was successfully applied to estimate the glucose concentration in human blood serum samples. Furthermore, the AuNP-ATP-cMWCNT was investigated for the non-enzymatic detection of hydrogen peroxide by using voltammetric and amperometric techniques, indicating a linear relationship in the range of 5 × 10-3to 13.10 mM. This journal is