Postmortem diagnosis of cantharidin-induced myocardial injury and postmortem interval estimation (PMI) are the challenges in forensic science. Cardiac biomarkers play an important role in the prediction and diagnosis of myocardial injury and can be used to determine the PMI. Based on the evidence, we aimed to explore the biomarkers which may be used for the postmortem diagnosis of cantharidin-induced myocardial injury and PMI estimation using the study of the proteins expression of TN-T, VEGF and HIF-1α by ELISA. Results of this study suggested that postmortem pathological changes were difficult to identify due to the autolysis of myocardium 72 h after death in cantharidin poisoning group. The plasma levels of TN-T and HIF-1α/TN-T are cardiac biomarkers with higher diagnostic accuracy for postmortem diagnosis of cantharidin-induced myocardial injury, VEGF/HIF-1α promises to be a biomarker for PMI estimation. Further studies are needed to verify these biomarkers, based on population, for being a useful tool in postmortem diagnosis of cantharidin-induced myocardial injury and PMI estimation.