Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method 1 have faster response time (<2 s) and lower detection limit (2.5±1.1 μM) compared to that prepared by Method 2 (response time: <5 sec and detection limit: 6.5±1.7 μM); glutamate sensors prepared by Method 2 have a larger linear detection range (20-352 μM) and higher sensitivity (86.8±8.8 nA·μM-1·cm-2, N=12) compared to those prepared by Method 1 (linear detection range: 20-217 μM and sensitivity: 34.9±4.8 nA·μM-1·cm-2, N=8). The applicability of the glutamate sensors in vivo was also demonstrated. The glutamate sensors were implanted into the rat brain to monitor the stress-induced extracellular glutamate release in the hypothalamus of the awake, freely moving rat.