Using two nano-porous pseudo carbon paste electrodes (Nano-PPCPEs) as the working electrodes, a saturated calomel electrode as the reference electrode, and a Pt electrode as the counter electrode to form a four-electrode system, a novel type of glutamate-acetylcholine electrochemical biosensor array was successfully fabricated. It was shown that the biosensor array displayed a high selectivity and sensitivity and the selective and simultaneous determination of glutamate and acetylcholine (ATCh) is feasible. For L-glutamate, a linear range from 5 x 10(-7) M to 1 x 10(-5) M with the detection limit of 2.5 x 10(-7) M was demonstrated, while for ATChl the linear range is from 5 x 10(-6) M to 2 x 10(-4) M with the detection limit of 1.5 x 10(-7) M. This research means a possibility to simultaneously detect more neurotransmitters with a nano-PPCPEs electrochemical biosensor array.