A reactor-separator combining immobilized penicillin acylase (IPA) and electrodialysis was developed for the production of 6-aminopenicillanicacid (6-APA)from the enzymatic hydrolysis of penicillin G (Pen-G) accompanying the continuous removal of by-product phenylacetic acid (PAA). The kinetics of Pen-G hydrolysis by IPA, the separation of PAA, 6-APA and Pen-G with electrodialysis, as well as the reaction-separation phenomena of Pen-G hydrolysis with IPA in the electrodialyzer were studied. The products inhibition mechanism and rate equation of Pen-G hydrolysis by IPA were obtained. The separation of PAA, 6-APA and Pen-G with electrodialysis was dependent on their molecular size and affinity toward the anion exchange membrane. The transport rates of three components decreased in the sequence PAA > 6-APA > Pen-G. Strong concentration polarization of Pen-G was observed in the vicinity of anion exchange membrane. An increase in Pen-G concentration enhanced the selectivity factor of PAA to 6-APA but reduced both the transport rates of PAA and 6-APA. The increase of current density promoted both the transport rates of PAA and 6-APA but reduced the selectivity of PAA to 6-APA. The utilization of electrodialysis was shown to have an improvement in the efficiency of Pen-G hydrolysis by IPA.