Cefprozil is an important semi-synthetic cephalosporin antibiotic. In this study, immobilized penicillin G acylase (PGA) is used to catalyze the acylation of 7- amino-3-(1-propenyl)-4-cephalosporanic acid (7-APRA) and 4-hydroxyphenylglycine methyl ester (HPGME) and a recyclable thermo-pH responsive PNB/PADB aqueous twophase system (ATPS) is used to synthesize cefprozil. In this system, the partition coefficient of cefprozil was 2.24 with 60 mmol/L (NH4)2SO4. In addition, the optimal enzymatic reaction conditions were found to be pH 6.5, 20°C, 78 u/mL immobilized PGA, 30 mmol/L 7-APRA and 90 mmol/L HPGME. In the PNB/PADB ATPS, the maximal yield of cefprozil was 75.81% with 60 mmol/L (NH4)2SO4 and in the single aqueous system the yield was 56.02%. The yields are thought to improve because there is a reduction in product inhibition.