Ethanol is the most widely-used psychoactive drug, which unregulated consumption causes damage on organs and the CNS, and lead to develop alcohol addiction, in which neurotransmitter systems are disrupted. Several studies investigated ethanol pharmacodynamics in CNS, yet an insufficiency of detailed knowledge remains. Because the optimal study of the fast ethanol concentration changes would require a rapid detection, we developed an implantable amperometric biosensor capable of real-time monitoring ethanol dynamics in brain ECF of animals. Different design of AOx-based biosensors were developed and characterized over a time of 28 days, in terms of apparent Michaelis-Menten parameters, sensitivity and efficiency to reject interfering electroactive molecules. Furthermore, the oxygen-, pH- and temperature-dependence of biosensors was studied. The best performing biosensor was implanted in vivo in the nucleus accumbens of freely moving rats, systemically administrated with ethanol. In addition, the effect of ranitidine was studied. The hydrogen peroxide, by-product of enzymatic ethanol oxidation, was electrochemically oxidized at the Pt transducer surface, generating an electrical signal telemetric recorded LIVE. Ethanol biosensor demonstrated to be a reliable tool to study the cause-and-effect relationships existing between alcohol and the neurotransmitter systems it affects, also providing a technique for the future testing of new drugs for the treatment of alcohol addiction.