BACKGROUND: Colorectal cancer (CRC) is a major global health burden. The current diagnostic tests have shortcomings of being invasive and low accuracy. AIM: To explore the combination of intestinal microbiome composition and multi-target stool DNA (MT-sDNA) test in the diagnosis of CRC. METHODS: We assessed the performance of the MT-sDNA test based on a hospital clinical trial. The intestinal microbiota was tested using 16S rRNA gene sequencing. This case-control study enrolled 54 CRC patients and 51 healthy controls. We identified biomarkers of bacterial structure, analyzed the relationship between different tumor markers and the relative abundance of related flora components, and distinguished CRC patients from healthy subjects by the linear discriminant analysis effect size, redundancy analysis, and random forest analysis. RESULTS: MT-sDNA was associated with . MT-sDNA and carcinoembryonic antigen (CEA) were positively correlated with the existence of , and alpha-fetoprotein (AFP) was positively associated with and . In the random forest model, the existence of , , , , , and can distinguish CRC from health controls. The diagnostic accuracy of MT-sDNA combined with the six genera and CEA in the diagnosis of CRC was 97.1%, with a sensitivity and specificity of 98.1% and 92.3%, respectively. CONCLUSION: There is a positive correlation of MT-sDNA, CEA, and AFP with intestinal microbiome. Eight biomarkers including six genera of gut microbiota, MT-sDNA, and CEA showed a prominent sensitivity and specificity for CRC prediction, which could be used as a non-invasive method for improving the diagnostic accuracy for this malignancy.