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Glutamate is a major excitatory neurotransmitter in the brain. It is involved in many normal physiological brain activities, but also neurological disorders and excitotoxicity. Hence, glutamate measurement is important both in clinical and pre-clinical studies. Pre-clinical studies often use amperometric biosensors due to their low invasiveness and the relatively small size of the devices. These devices also provide fast, real-time measurements because of their high sensitivity. In the present study, diethylene glycol (DEG), neopentyl glycol (NPG), triethylene glycol (TEG), and glycerol (GLY) were used to increase the long-term stability of glutamate biosensors. The evaluation was made by measuring variations of the main enzymatic (V_MAX and K_M) and analytical (Linear Region Slope (LRS)) parameters. Of the glycols tested, TEG was the most promising stabilizer, showing about twice as high V_MAXmaintained over a greater duration than with other stabilizers tested. It is also yielded the most stable linear region slope (LRS) values over the study duration. Moreover, we highlighted the ability of glycols to interact with enzyme molecules to form a containment network, able to maintain all the layered components of the biosensor adhering to the transducer.