Objective: To investigate the methods and experimental conditions of constructing myocardial endoplasmic reticulum stress (ERS) model in vitro. Methods: Langendorrf perfusion device was used to make rat heart ischemia/reperfusion model in vitro, and left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR) and the maximum rate of change of left ventricular pressure (+/-LVdp/dtmax) were continuously monitored by the PowerLab system. The expression of ERS classical marker glucose-regulated protein (GRP) 78 in myocardium was detected by western-blot analysis after ischemia (stop perfusion) different times (30min, 35min, 40min and 45min) / reperfusion 120min, the expression of C/EBP homologous protein(CHOP) was detected, too, and the mRNA level of GRP 78 and CHOP were detected by reverse transcription-polymerase chain reaction（RT-PCR）; myocardial tissue slices were incubated in vitro and treated by ERS stimulant tunicamycin (Tm) or dithiothreitol (DTT) at different concentrations for 3h and 6h, respectively, and the expression of GRP78 and CHOP were detected by western-blot. Results: The highest expression of GRP78 protein was detected in myocardium from the isolated perfused rat heart with ischemia 40min / reperfusion 120 min(P <0.01), CHOP protein、GRP78 mRNA and CHOP mRNA increased significantly（P <0.01，P <0.05和P <0.05） and at the same time, compared with control group, HR, ±LVdp/dtmax, coronary perfusion flow(CPF), and left ventricular pressure(△LVP, the difference between LVESP and LVEDP) decreased, and LVEDP increased (all P <0.01) . Compared with control group, myocardial tissue slices incubated with Tm / DTT for 3h, GRP78 in Tm10μg/mL group and DTT 2 mmol/L group significantly increased (both P <0.001); GRP78 in DTT 10 mmol/L group increased (P <0.05), but not as much as that in DTT 2 mmol/L. The expression of CHOP increased significanly, too（P <0.05 and P <0.01）. Conclusion: Using both isolated rat heart ischemia/reperfusion and myocardial tissue slices incubation methods, the in vitro myocardial ERS model can be successfully constructed.