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Background. Colorectal cancer (CRC) is one of the most common cancers in the world, identification of biomarkers for early detection of CRC represents a relevant target. The present study aims to determine serum peptidome patterns for CRC diagnosis. Methods. The present work focused on serum proteomic analysis of 32 health volunteers and 38 CRC by ClinProt Kit combined with mass spectrometry. This approach allowed the construction of a peptide patterns able to differentiate the studied populations. An independent group of serum (including 33 health volunteers, 34 CRC, 16 colorectal adenoma, 36 esophageal carcinoma, and 31 gastric carcinoma samples) was used to verify the diagnostic and differential diagnostic capability of the peptidome patterns blindly. An immunoassay method was used to determine serum CEA of CRC and controls. Results. A quick classifier algorithm was used to construct the peptidome patterns for identification of CRC from controls. Two of the identified peaks at m/z 741 and 7772 were used to construct peptidome patterns, achieving an accuracy close to 100% (>CEA, P < 0.05). Furthermore, the peptidome patterns could differentiate validation group with high accuracy. Conclusions. These results suggest that the ClinProt Kit combined with mass spectrometry yields significantly higher accuracy for the diagnosis and differential diagnosis of CRC.