A newly bienzymatic glucose biosensor was prepared for selective and sensitive detection of glucose. This biosensor was built by simultaneous immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) into the gold nanoparticles-mesoporous silica composite (GNPs-MPS) matrix, and then casting the composite on glassy carbon electrode (GCE) using Nafion as a cross-linker. Cyclic voltammetry (CV) and amperometry were employed to investigate the catalytic behavior of the biosensor to the oxidation of glucose. As a result, the biosensor exhibited an excellent bioelectrocatalytic response to glucose, as well as good long-term stability and reproducibility.