An electrochemical aptamer-based sensor was developed for glutamate, the major excitatory neurotransmitter in the central nervous system. Determining glutamic acid release and glutamic acid levels is crucial for studying signal transmission and for diagnosing pathological conditions in the brain. Glutamic acid-selective oligonucleotides were isolated from an ssDNA library using the Capture-SELEX protocol in complex medium. The selection permitted the isolation of an aptamer 1d04 with a dissociation constant of 12 µM. The aptamer sequence was further used in the development of an electrochemical aptamer sensor. For this purpose, a truncated aptamer sequence named glu1 was labelled with a ferrocene redox tag at the 3'-end and immobilized on a gold electrode surface via Au-thiol bonds. Using 6-mercapto-1-hexanol as the backfill, the sensor performance was characterized by alternating current voltammetry. The glu1 aptasensor showed a limit of detection of 0.0013 pM, a wide detection range between 0.01 pM and 1 nM, and good selectivity for glutamate in tenfold diluted human serum. With this enzyme-free aptasensor, the highly selective and sensitive detection of glutamate was demonstrated, which possesses great potential for implementation in microelectrodes and for in vitro as well as in vivo monitoring of neurotransmitter release.