Objective To purify AmpC beta-lactamase from Enterobacter cloacae and study its physical and chemical characteristics. Methods The bacteria were fragmentized by ultrasonication and AmpC beta-lactamase was purified by ion-exchange. The properties of the purified enzyme,such as molecular weight and isoelectric point were determined by electrophoresis. Results The purified AmpC beta-lactamase was obtained and its specificity to substrate was sufficient for classification of class C cephalosporinase. Conclusion AmpC can be successfully purified from Enterobacter cloacae and is effective for application.