Cefprozil, a semi-synthetic antibiotic, has been used in pediatric therapy. The purpose of this study was to improve the yield of cefprozil and obtain purer product in an aqueous two-phase system composed of P-ADB and P-MDB. P-ADB and P-MDB were synthesized using acrylic acid, methacrylic acid, 2-dimethylaminoethyl methacrylate, and butyl methacrylate. Cefprozil was synthesized with 7-amino-3-(1-propenyl)-4-cephalosporanic acid (7-APRA) as the main ring, (R)-amino-(4-hydroxyphenyl) acetic acid methyl ester hydrochloride (D-HPGME-HCl) as the acyl donor and the immobilized penicillin acylase as the catalyst. 7-APRA and D-HPGME-HCl were concentrated in the P-MDB-rich phase, while cefprozil was in the P-ADB-rich phase. The optimal partitiona coefficient of cefprozil was 1.84 with 40 mM KSCN, while the enzyme reaction yield of cefprozil reached 99.39% compared with 77.61% in the aqueous-phase system. The recovery of the two copolymers was up to 96.01% at pH 3.54. After crystallization, the purity of the product reached 88.02%, and no copolymer remained in the product. The combination of cefprozil DMF was detected by gas chromatography-mass spectrometry, FT-IR spectroscopy, thermogravimetric analysis, and electrospray ionization mass spectrometry. The results showed that no chemical bond existed between cefprozil and DMF.