Drug-induced cardiotoxicity is a leading cause of failure in drug development and predicting its occurrence in non-clinical studies is the primary preventive measure. The present study aimed to evaluate the changes in biomarkers during acute and chronic myocardial injury induced by doxorubicin (DOX) in rats. A rat model of acute myocardial injury was established through a single-dose, intraperitoneal injection of DOX (40 mg/kg), the changes in biomarkers were measured at 2, 4, 8 and 24 h after administration, following DOX administration, creatine kinase (CK) and fatty acid-binding protein 3 (FABP3) levels increased between 8 and 24 h, whereas cardiac troponin I (cTnI) peaked at 8 h. To establish a chronic myocardial injury model, rats received 1, 2 or 3 mg/kg DOX weekly by caudal vein injection for 2, 4, 6 or 7 weeks, the changes in biomarkers were detected at 2, 4, 6 and 8 weeks, the results showed that cTnI increased significantly after 2 and 8 weeks of administration. A significant increase in FABP3 and microRNA (miR)-146b levels was observed after 8 weeks of administration. Receiver operating characteristic curve and correlation analysis showed that cTnI and miR-146b had relatively high predictive values for chronic myocardial injury (area under the curve, 0.83 and 0.71, respectively) and were closely correlated with myocardial damage. These data suggested that CK, cTnI and FABP3 were relatively sensitive to DOX-induced acute myocardial injury, whereas cTnI and miR-146b were relatively sensitive to DOX-induced chronic myocardial injury.