To increase the sensitivity and specificity and decrease the cost on determining the isoenzymes of creatine kinase (CK), including CK-MM, CK-MB, and CK-BB, we used the electrophoresis bath with different buffers to isolate the isoenzymes and used the fluorescent scanner to determine the content of each isoenzyme. We found that the concordance appeared in sensitivity and specificity in comparison with the MOPS electrophoretic system. There was a positive correlation between our method and MOPS, and the cost was much less than that of the MOPS.