Abstract: Objective To compare the different activity and quality of serum creatine phosphokinase isoenzyme （CK-MB） in healthy children at different ages and to study its rational clinical application. Methods A total of 184 healthy children were divided into different age groups and 30 healthy adults served as a control group. Activity and quality of their serum CK-MB were detected by inhibition enzyme immunoassay and double antibody sandwich enzyme-link immunoassay, respectively. Results A positive correlation was observed between the activity and quality of serum CK-MB in the different groups. In children group, the activity and quality CK-MB decreased with the increasing age, almost reaching those in the adults. The CK-MB quality was normal in the different age groups. However, the CK-MB activity was slightly higher than normal in children at a younger age. Conclusion It is necessary to consider the age when the Clinician determines the condition of patients based on the CK-MB activity and quality. Moreover, CK-MB quality assessment is better than the activity assessment in clinical diagnosis of patients.