In this work, pristine graphene was produced through a novel single step exfoliation of graphite in mild sonochemical alcohol-water treatment. The developed green synthesis approach successfully eradicates issues associated with conventional methods which use organic solvents, acids and oxidizers, leaving undesirable functional groups attached to the graphene surface. Results from cyclic voltammetry and amperometric analysis showed a wide linear range up to 5 mM and sensitivity improvements of more than 22 times in comparison to the control sample. Subsequently, an electrochemical glucose biosensor was fabricated by the immobilization of glucose oxidase (GOx) via bi-functional linkers. This reliable surface modification method provides irreversible non-covalent bonding between graphene and the enzymatic amide groups, while preserving the sp2 graphene structure, whilst promoting better electron transfer kinetics between the FAD/FADH2 redox sites of GOx at the modified electrode surface. The fabricated biosensor exhibited satisfactory long-term stability, reproducibility and high selectivity for glucose detection and showed significant improvements when compared to unmodified electrodes.