We have developed an amperometric microbiosensor for real time monitoring L-glutamate release in neural tissue, based on enzymatic oxidation catalyzed by the L-glutamate oxidase. By means of a sol-gel coating method, L-glutamate oxidase was entrapped in a biocompatible gel layer that provided a benign environment and retained enzyme activity on the surface of Pt microelectrode. Prior to gel layer formation, a modification on the surface of Pt microelectrode with poly(phenylene diamine) enabled the microbiosensor screen majority of common potential interfering substances existing in physiological samples. The miniaturized biosensor achieved a steady state response to l-glutamate within 10 s and exhibited a linear dependence on the concentration of L-glutamate from 0.5 to 100 micromol L(-1) with a high sensitivity of 279.4 +/- 2.0 microA (mmol L(-1))(-1) cm(-2) (n = 4, R.S.D. = 2.8%). The microbiosensor also exhibited excellent long-term stability in dry storage. We have successfully used the microbiosensor for real time measuring of L-glutamate in vivo.