The separation behavior of phenylacetic acid (PAA), 6-aminopenicillanic acid (6-APA), and penicillin-G (Pen-G) with electrodialysis under constant voltage was studied. The effects of ionic concentrations and electric field strength on their separation behavior were investigated. The sorption of PAA, 6-APA, and Pen-G in the anion-exchange membrane, and the variations of current and current efficiencies with time during electrodialysis, were also examined. The electrodialysis of PAA, 6-APA, and Pen-G was closely dependent on their ionic size and their affinity toward the anion-exchange membrane. 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. The concentration polarization of Pen-G in the vicinity of anion-exchange membrane severely retarded the transport of PAA and 6-APA, and became more serious with the increase of Pen-G concentration. The increase of electric field strength accelerated the transport of PAA, 6-APA, and Pen-G. However, more serious concentration polarization of Pen-G occurred and the separation ratio of PAA to 6-APA decreased.