Objective To evaluate an improved modification of TTC staining method for measuring myocardial infarct size after ischemia-reperfusion in rats. Methods Twenty healthy SPF male 8-week-old SD rats were randomly divided into two groups: Group A with conventional TTC staining, and group B with the modified TTC staining method for measuring myocardial ischemia-reperfusion injury. The infarct size was caculated and the serum cTnI levels were determined. Results The infarcted myocardium was well detected in both groups A and B. There were no significant differences in the myocardial infarct sizes measured in the groups A and B (48.69±5.37 % vs. 47.41±3.28%, P>0.05). There were no significant differences in the serum cTnI levels assayed in the groups A and B (4.51±0.88 ng/mL vs. 4.70±0.71 ng/mL, P>0.05). But compared with the group A, the color contrast of stained myocardial slice and the distinguishing infarction area and non-infarction area were much clearer in the group B. Conclusions Our modified TTC staining technique using in vivo staining is an economic, convenient, fast and efficient method, being easy to control, time-saving and inexpensive, and enhances the staining effect in evaluating the size of myocardial ischemia/reperfusion injury more accurately.