A hybrid flow injection-nanobiosensor system for the detection of glucose at low potential is presented in this paper. For the fabrication of the biosensor, highly ordered gold nanowire arrays (AuNWAs) are synthesized by direct electrodeposition in combination with template approach. Glucose oxidase and horseradish peroxidase are immobilized on the surface of the resultant AuNWAs by cross-linking with glutaraldehyde (GLA) and bovine serum albumin (BSA). In combination with a flow injection system, the biosensor demonstrate high performance in glucose detection with a sensitivity of 25.34 μA. cm-2. mM-1, a linear range between 5 and 1000 μM, a detection limit of 1 μM, and a sample throughput of 45 h-1. High selectivity against uric acid, nitrate and chloride ions is also achieved. In addition, it shows good reproducibility, repeatability and stability, which are beneficial for the practical applications in glucose detection. Finally, the developed nanobiosensor is applied to the detection of glucose in river water and the biosensor presents good performance with a relative standard deviation of 1.33% and a recovery rate of 100.4∼102.9%.