A glutamate biosensor based on cross‐linked glutamate oxidase with chitosan (CHIT) on a carbon nanotube (CNT)‐modified gold nanowire (AuNW) was constructed and applied in a flow injection analysis (FIA) system. The AuNW was produced using an alumina template membrane and characterized using scanning electron microscopy (SEM) with a 120‐nm diameter. The CHIT cross‐linked CNT slurry was mixed with AuNW and cast on the surface of a glassy carbon electrode (GCE), followed by immobilization with the glutamate oxidase. Optimization of the experimental parameters, including the applied potential, enzyme loading, flow rate, type of buffer and pH, resulted in values of 0.7 V, 30 μL, 1.0 mL min⁻¹, phosphate buffer, and pH 7.4, respectively. The analytical working curve was linear in the ranges from 10 to 100 (R²=0.9981) and 100 to 20,000 μM glutamate (R²=0.9821) with a low detection limit of 1.2 μM (S/N=3). A glutamate biosensor with an FIA system was used to determine glutamate in samples of Thai commercial food products.