Objective To investigate the value of serum creatine kinase isoenzyme(CK-MB), cardiotrophin-1(CT-1) and heart fatty acid binding protein(H-FABP) in determining myocardial injury after neonatal asphyxia. Methods From January 2018 to December 2019, 98 cases of neonatal asphyxia(observation group), including 31 cases with myocardial injury, and 50 cases of healthy newborns were selected as the control group. The serum CK-MB, CT-1 and H-FABP levels were detected. Results There was no significant difference in gender, gestational age, body weight, and length between the observation group and the control group(P＞0.05). The Apgar score(5.30±0.82) of the observation group at 5 min after birth was significantly lower than that of the control group(9.40±0.32)(P＜0.05). The serum CK-MB, CT-1 and H-FABP in the observation group were(6.62±1.31) ng/mL,(68.46±18.84) ng/L and(21.03±8.11) ng/mL, which were significantly higher than those in the control group(P＜0.05). The serum CK-MB, CT-1 and H-FABP in children with myocardial injury of the observation group were(7.01±1.22) ng/mL,(78.11±12.03) ng/L and(30.45±9.54) ng/mL, which were significantly higher than those of children without myocardial injury(P＜0.05); The area under ROC curve of serum CK-MB, CT-1 and H-FABP in diagnosis of myocardial injury was 0.629, 0.815 and 0.862, respectively(P＜0.05). The area under ROC curve of CT-1 and H-FABP was higher than that of CK-MB(P＜0.05). Conclusion CK-MB, CT-1 and H-FABP have certain application value in judging myocardial injury after neonatal asphyxia, among which CT-1 and H-FABP have higher value.