7-aminocephalosporanic acid (7-ACA) is the key intermediate for the synthesis of semisynthetic cephalosporin antibiotics and enzyme cephalosporin-C acylase (CPC acylase) plays an important role in the conversion of cephalosporin-C to 7-ACA. With an aim to increase the yield of 7-ACA production by Micrococcus luteus, a stepwise strategy, statistical medium was applied for optimizing the medium composition for the production of CPC acylase. Purified enzyme was found to be of 80 kDa. The optimum pH and temperature for the production of 7-ACA were 7.6 and 340C, respectively. The Km and Vmax were estimated to be 9.43 mg/mL and 7.65 U/mL, respectively.