Although intramyocardial hemorrhage (IMH) is a poor prognostic factor caused by ischemia reperfusion injury, little evidence is available regarding the association between IMH volume and biomarkers. In the present study, we measured IMH volume using three-dimensional (3D) T1-weighted magnetic resonance imaging (T1-MRI) and investigated its association with biomarkers. Moreover, the accuracy of semi-automatic measurement of IMH volume was validated. We retrospectively enrolled 33 consecutive patients (mean age 67 ± 11 years) who underwent cardiac MRI after reperfusion therapy for acute myocardial infarction. IMH was observed in 4 patients (12.1%). Receiver operating characteristics (ROC) analysis of creatine kinase (CK) and CK-muscle/brain (CK-MB) tests for detecting IMH were performed. IMH volume measured using semi-automatic methods by a 2 standard deviation (SD) threshold was compared to manual measurements using the Spearman’s correlation coefficient (ρ) and Bland–Altman analyses. ROC analysis revealed optimal cutoff values of CK: 2460 IU/l and CK-MB: 231 IU/l (area under the curve: 0.95 and 0.91; sensitivity: 86% and 79%; specificity: 100% for both). IMH volume with the 2SD threshold correlated with that of the manual measurement [5.84 g (3.30 to 9.00) g vs. 8.07 g (5.37 to 9.33); ρ: 0.85, p < 0.01; bias (limit of agreement): − 0.01 g (− 0.51 to 0.49); intraclass correlation coefficients 0.84 (0.75 to 0.90)]. Our findings could help identify the risk of IMH after reperfusion therapy with biomarkers. 3D T1-MRI can semi-automatically provide accurate IMH volume without being time-consuming.