A novel colorimetric glucose biosensor based on graphene oxide (GO)-gold nanoparticles (AuNPs)-glucose oxidase (GOD) is fabricated using chemical cross-linking and electrostatic methods. GOD catalyzes the glucose oxidation to produce H₂O₂, which in turn shifts the local surface plasmon resonance (LSPR) band of GO-AuNPs to achieve glucose detection. GO improves the stability of composite particles, reduces metal particle biotoxicity, and enhances sensor response. The microstructure and optical properties of the composite particles are characterized using transmission electron microscope (TEM), Raman spectroscopy, and UV-Vis spectrophotometer. The optimal detection conditions of the sensor are systematically investigated, and the sensor exhibited high sensitivity and selectivity for tested glucose mass concentrations. The sensitivity of the sensor is 14 nm/(mg/mL) in the range of 0-1.6 mg/mL, which suggests that this sensor can be used for glucose detection in clinical settings. Our sensor combines the advantages of GO and AuNPs, and holds potential for simple, stable, and cost-effective use for glucose detection.