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Abstract Background Tumor pyruvate kinase M2 isoform (tM2-PK), which is an isoform of PK-glycolytic enzyme and appears on the surface of cancerous proliferating cells, has been used as a diagnostic biomarker for colorectal cancer (CRC). The aim of this study was to evaluate the tM2-PK measurement test for the diagnosis of CRCs and adenomatous polyps in plasma and stool samples in an Iranian population. Methods In this prospective study, a total of 226 stool and 178 plasma samples were received from patients referred to colonoscopy units. tM2-PK enzyme was measured using two separate ScheBo-Biotech-AG ELISA kits for stool and plasma samples. Results According to ROC curves, in the tumor group, at the cut-off value of 4 U/ml, the sensitivity of fecal tM2-PK test was 100% and the specificity was 68%, and in the polyp group, the sensitivity and specificity were 87 and 68%, respectively. For tumor detection in plasma specimens, a cut-off value > 25 U/ml has a sensitivity and specificity of 90.9 and 91.3%, respectively. Similarly, for polyp detection, a cut-off value > 19 U/ml has a sensitivity of 96.3% and the specificity of 85.5%. Conclusions Based on our results, a cut-off range of 4.8–8 U/ml and >  8 U/ml could be used to detect polyp and tumor in stool samples, respectively. Similarly, a cut-off range of 19–25 U/ml and > 25 U/ml is recommended in plasma samples, suggesting tM2-PK test as a non-invasive assay to diagnose CRC and adenomatous polyps.