A novel enantioselective potentiometric sensor was constructed based on optical active polyaniline (PAn) films. The sensor with high stability showed discrimination behavior toward one isomer of racemic phenylalanine (Phe). The (-) or (+) PAn-based electrodes exhibited an excellent Nernstian slope of approximately 60 mV per decade for L-Phe or D-Phe, respectively. In addition, when the sensor was exposed to the mixture of enantiomers, the slope of the response changed along with their proportions, which would be employed as an index of the enantiomeric purity of the mixture. At last, the mechanism of chiral discrimination for Phe was discussed.