Engineering of nanoparticle (NP) surfaces offers an effective approach for the development of enzymatic biosensors or microbial fuel cells with a greatly enhanced direct electron transport process. However, lack of control over the surface functionalization process and the operational instability of the immobilized enzymes are serious issues. Herein, we demonstrate a facile and green deep eutectic solvent (DES)-mediated synthetic strategy for efficient amine-surface functionalization of silicon dioxide and to integrate small gold nanoparticles (AuNPs) for a glucose biosensor. Owing to the higher viscosity of the DES, it provides uniform surface functionalization and further coupling of the AuNPs on the SiO2 support with improved stability and dispersion. The amine groups of the functionalized Au-SiO2NPs were covalently linked to the FAD-center of glucose oxidase (GOx) through glutaraldehyde as a bifunctional cross-linker, which promotes formation of "electrical wiring" with the immobilized enzymes. The Au-SiO2NP/GOx/GC electrode exhibits direct electron transfer (DET) for sensing of glucose with a sensitivity of 9.69 μA mM-1, a wide linear range from 0.2 to 7 mM and excellent stability. The present green DES-mediated synthetic approach expands the possibilities to support different metal NPs on SiO2 as a potential platform for biosensor applications.