Objectives: This study aimed to evaluate the early and rapid diagnostic value and the cut-off value of Heart-type fatty acid-binding protein (H-FABP) for the perioperative myocardial infarction (PMI) diagnosis after off-pump coronary artery bypass grafting (OPCABG) surgery. Method: 109 patients scheduled for first-time, elective, and isolated OPCABG surgery were erolled in this single-center, prospective and consecutive study. Serial samples for measurement of H-FABP at pre-anesthesia induction (T0, baseline), completion of last distal anastomosis (T1), on ICU admission (T2), and at 2h (T3), 4h (T4), 8h (T5), 24h (T6), and 48h (T7) were taken to analyze for H-FABP. CK-MB and cTnI were taken at T4, T5, T6, and T7. Patients were first divided into three groups according to the level of cTnI and ECG changes within the early 24 hours. Results: 44 patients served as a control group, 52 patients identified as myocardial injury group, and 13 patients defined as myocardial infarction group. The level of H-FABP released was significantly higher in the myocardial infarction group than another two groups. Using a cut-off value of 20.53 ng/ml for H-FABP, the sensitivity was 84.62%, 92.31%, and 76.92% immediately, 2 hours, and 4 hours after entering the ICU. The sensitivity of combined H-FABP and cTnI was 92.31% and 84.62% at 4 and 8 hours after entering the ICU. Conclusions: With an optimal H-FABP cut-off level of 20.53 ng/ml, H-FABP was the early and reliable biomarker for PMI diagnosis after OPCABG.