An agarose gel electrophoretic method for separating creatine kinase (CK) isoenzymes and its isoforms(CK-MB and CK-MM isoforms)simultaneously was established.It is based on a suitable discontinuous buffer system.By passing a larger current through the gel under a lower applied voltage, the separating time was shortened to 30 min. The measurements of CK-MB%,CK-MB₁%,CK-MB₂%,CK-MM₁%,CK-MM₂%,CK-MM₃% can be completed within one hour. The method is sufficinently sensitive to detect the CK-MB isoenzyme and CK-MM isoforms at the concentration of lowest detection limit 2.2U/L and 32U/L respectively. Thus, this system can be used to rapidly, sensitively and precisely quautify the isoenzymes and isoforms of CK.