A mediatorless glucose biosensor was developed by the immobilization of glucose oxidase (GOx) to graphene-functionalized glassy carbon electrode (GCE). The surface of GCE was functionalized with graphene by incubating it with graphene dispersed in 3-aminopropyltriethoxysilane (APTES), which acted both as a dispersion agent for graphene and as an amine surface modification agent for GCE and graphene. This was followed by the covalent binding of GOx to graphene-functionalized GCE using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) based crosslinking. Graphene provided signal enhancement by providing greater surface area for GOx binding, while APTES-functionalization led to a higher GOx immobilization density by providing free amino groups for crosslinking. The developed biosensor used a redox potential of -0.45 V (vs. Ag/AgCl) for detecting glucose in the diabetic pathophysiological range 0.5-32 mM. There was no interference from endogenous electroactive substances and drug metabolites. The developed biosensor was further validated for detecting blood glucose in commercial artificial blood glucose linearity standards in the range 1.4-27.9 mM. Therefore, it is ideal for diabetic blood glucose monitoring. The developed bioanalytical procedure for preparation of GOx-bound graphene-functionalized GCEs had high production reproducibility and high storage stability, which is appropriate for the commercial mass production of enzyme-bound electrodes.