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<h4>Background</h4>This study aimed to evaluate the association between intraoperative blood loss and myocardial injury after non-cardiac surgery (MINS), which is a severe and common postoperative complication.<h4>Methods</h4>We compared the incidence of MINS based on significant intraoperative bleeding, defined as an absolute hemoglobin level < 7 g/dL, a relative hemoglobin level less than 50% of the preoperative measurement, or need for packed red cell transfusion. We also estimated a threshold for intraoperative hemoglobin level associated with MINS.<h4>Results</h4>We stratified a total of 15,926 non-cardiac surgical patients with intraoperative hemoglobin and postoperative cardiac troponin (cTn) measurements according to the occurrence of significant intraoperative bleeding; 13,416 (84.2%) had no significant bleeding while 2,510 (15.8%) did have significant bleeding. After an adjustment with inverse probability weighting, the incidence of MINS was higher in the significant bleeding group (35.2% vs. 16.4%; odds ratio, 1.58; 95% confidence interval, 1.43-1.75; p < 0.001). The threshold of intraoperative hemoglobin associated with MINS was estimated to be 9.9 g/dL with an area under the curve of 0.643.<h4>Conclusion</h4>Intraoperative blood loss appeared to be associated with MINS. Further studies are needed to confirm these findings.<h4>Clinical registration</h4>The cohort was registered before patient enrollment at https://cris.nih.go.kr (KCT0004244).