Although serum troponin level is the gold standard under the universal definition of acute myocardial infarction (AMI), serum creatinine kinase (CK) and creatine kinase-myocardial band (CK-MB) is still measured in clinical practice as the compliment of troponin level. The purpose of this retrospective study is to illustrate the dramatic change of CK-MB/CK ratio by comparing CK-MB/CK ratio in patients with ST-segment elevation myocardial infarction (STEMI) among ≤ 24 h before reaching peak CK, peak CK, ≤ 24 h after reaching peak CK, and 24–48 h after reaching peak CK. We included 502 patients with STEMI. We calculated each average CK-MB/CK ratio at ≤ 24 h before reaching peak CK, peak CK, ≤ 24 h after reaching peak CK, and 24–48 h after reaching peak CK. The average values were compared using Friedman test. The average CK-MB/CK ratio at ≤ 24 h before reaching peak CK, peak CK, ≤ 24 h after reaching peak CK, and 24–48 h after reaching peak CK was 0.096 (9.6% of CK), 0.098 (9.8% of peak CK), 0.076 (7.6% of CK), and 0.028 (2.8% of CK), respectively. The Friedman test suggested that the CK-MB/CK ratio significantly declined after reaching peak CK (p < 0.001). In conclusion, the CK-MB/CK ratio was around 0.1 (10% of CK) until CK-MB and CK reached the peak, but dropped sharply after reaching peak CK. The CK-MB/CK ratio less than 0.1 (10% of CK) cannot be used to rule out the possibility of AMI, when the onset of symptom is unclear or late presentation.