We report the first on-line electrochemical sensor for the continuous measurement of γ-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Os-poly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the α-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/μM for GABA under optimized conditions and shows almost no response when 10 μM glutamate is continuously injected. A detection limit of 0.1 μM is obtained with a linear range of 0.1−10 μM. GABA can be measured in the absence of α-ketoglutarate when there is l-glutamate in the sample solution, which is a typical ...