We report an enzyme-based potentiometric glutamate sensor combined with a semiconductor device for neurotransmitter imaging, the response of which is based on the detection of the interfacial potential of a gold electrode produced by redox and enzyme reactions. First, we confirmed that the interfacial potential increased logarithmically with the increasing concentration ratio of hexacyanoferrate(III) to hexacyanoferrate(II). The proposed sensor could detect the interfacial potential change of the gold electrode with a slope of 66.1 mV/decade. H2O2 and L-glutamate were detected as a function of the change in the interfacial potential of the gold electrode in the range of 0.01–1 mM. Slopes of 55.2 and 41.9 mV/decade were obtained as the sensitivities for H2O2 and L-glutamate, respectively. These estimated values are larger than the theoretical value of 29.5 mV/decade. Although the reason why the difference between the estimated value and the theoretical value occurred could not be revealed, the interfacial potential is expected to depend on some kind of reaction related to H2O2 concentration because the logarithmic response was obtained for H2O2 concentration. Therefore, the results suggested that the sensor can detect the L-glutamate concentration.