Overoxidized polypyrrole films templated with L-glutamatehave been utilized for enantioselective detection of L- andD-glutamic acid. Various important fabrication factorscontrolling the performance of the overoxidized polypyrrole films have been investigated using fluorescencespectrometry in conjugation with the electrochemicalquartz crystal microbalance technique. The measuredfluorescence intensity was related to the concentration ofglutamate taken up into the films. It was found thatL-glutamate was inserted ~30 times higher into the filmthan D-glutamate. Several key parameters such as appliedpotential and pH of amino acid solution were varied toachieve the optimum sensor response. The sensor templated with L-glutamic acid also exhibited excellent selectivity over several other L- and D-amino acids. Higherenantioselectivity of overoxidized polypyrrole film thanthat for previously reported imprinted polymers can beattributed to the potential-induced uptake/release oftargeted molecules.