Penicillin G acylase from Alcaligenes faecalis(AfPGA) was used for the biosynthesis of cephalosporins. Optimization of the reaction was accomplished and the optimal results were achieved in phosphate buffer of pH 7.0 at 25 ℃, with substrate concentration of 60 mmol/L and enzyme of 3 U/mL. Conversion of substrate reached 85%. The results proved that AfPGA was eximious on enzyme activity. Isolation method was studied and resin was applied for separation. The purity of product reached 95% and the main by-product was less than 0.5%. The results were according with the pharmacopoeia and made the biotransformation process possible for industrial application.