Since early diagnosis is very important for treating CRC, we decided to detect peripheral serum canopy fibroblast growth factor signaling regulator 2 (CNPY2) isoform 2 to verify its diagnostic value for CRC patients. Serum samples were collected from 430 CRC patients and 201 healthy controls. Enzyme-linked immunosorbent assay (ELISA) detection kits for CNPY2 isoform 2 were generated and then applied to measure serum CNPY2 isoform 2 concentrations. Serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were also measured. The median serum CNPY2 isoform 2 concentrations in all CRC patients were significantly higher than those in the healthy control group (all P<0.001). Those with stage I CRC presented the highest area under the receiver operating characteristic curve (AUC) for CNPY2 isoform 2 [0.707, 95% confidence interval (CI): 0.649–0.765, P<0.001]. The diagnostic efficiency of the combination of CNPY2 isoform 2, CEA and CA19-9 was significantly higher than that of each biomarker detected separately (all P<0.0167). Serum CNPY2 isoform 2 may be a valuable biomarker for the early detection of CRC and presents an improvement in the diagnostic efficiency by combination of CEA and CA19-9.