The separation behavior of phenylacetic acid (PAA), 6-aminopenicillanic acid (6-APA) and penicillin-G (Pen-G) with electrodialysis under constant current was studied. The effects of ionic concentrations and current density on their separation behavior were investigated. The sorption of PAA, 6-APA and Pen-G in the anion exchange membrane, and the variations of applied voltage and current efficiencies with time during electrodialysis were also examined. It was found that the molecular size and the affinity toward the anion exchange membrane played a key role for the electrodialysis of PAA, 6-APA and Pen-G. Phenylacetic acid had the lowest affinity toward the membrane but the fastest transport rate. Penicillin-G had the highest affinity toward the membrane but the slowest transport rate. With the increase of Pen-G concentration, the concentration polarization of Pen-G in the vicinity of anion exchange membrane became very serious, which severely retarded the transport of PAA and 6-APA. Although the increase of current density accelerated the transport of PAA, 6-APA and Pen-G, more serious concentration polarization of Pen-G occurred and the separation ratio of PAA to 6-APA decreased.