To investigate the clinical value of serum markers of colorectal cancer (CRC) and fecal occult blood testing (FOBT) in patients with CRC and the relationship between these biomarkers.Methods Stool and blood markers were collected from a total of 7, 537 patients from Nanjing Medical University Affiliated Cancer Hospital from January 2001 to December 2021, and the diagnostic value of these markers and their relationship with clinicopathological features were retrospectively collected.Results The median levels of cfDNA, serous CEA and CA19-9 in patients with colon cancer were 17.32 ng/ml, 3.59 ng/ml and 15.63 U/mL, respectively.The median levels of the three biomarkers in patients with rectal cancer were 15.61ng/ml, 3.15 ng/ml and 12.9 U/ml, respectively.Plasma cfDNA, serum CEA and CA 19-9 levels in colon cancer were remarkably higher than those in rectal cancer.The positive rates of cfDNA, FOBT, CEA and CA19-9 were 67.90%, 39.37%, 48.67% and 23.35% respectively.The sensitivities of cfDNA, FOBT, CEA and CA19-9 were 56.5%, 84.6%, 49.9% and 59.9%, respectively, with specificities 58.6%, 32.3%, 84.0% and 95.5%, respectively.Positive FOBT and CEA significantly correlated with gender, age and tumor location of CRC patients. Likewise, positive serum cfDNA and CA19-9 were markedly correlated with age and tumor location.Joint detection of these makers could significantly improve the positive rates.Furthermore, the fecal marker FOBT was not correlated with the serum marker CEA, CA19-9and cfDNA significantly.Conclusion CfDNA has a high positive rate in colorectal cancer, and combined detection of these indicators can improve the detection rate of colorectal cancer.Moreover, our findings indicated that there were differences in the relation between these markers and CRC clinical parameters