Colorectal cancer is one of the most frequent causes of cancer-related deaths worldwide. Thus, there is a need for timely diagnosis and effective treatment. Our aim in the present study was to detect the serum level of trefoil factor 3 protein and evaluate the diagnostic accuracy of trefoil factor 3 in patients with colorectal cancer. We collected serum samples from 204 participants (127 patients with colorectal cancer, 35 patients with polyps, and 42 healthy controls). The levels of serum trefoil factor 3 and carcinoembryonic antigen expression were measured by enzyme-linked immunosorbent assay. Receiver operating characteristic curves were plotted to calculate the diagnostic accuracy of trefoil factor 3 in patients with colorectal cancer. The serum levels of trefoil factor 3 in patients with colorectal cancer (6.66 ± 2.4 ng/mL; P < .00l) and polyps (3.86 ± 1.3 ng/mL; P < .00l) were significantly increased compared to healthy controls (2.09 ± 1.0 ng/mL). Moreover, the area under the receiver operating characteristic curve for trefoil factor 3 was greater than carcinoembryonic antigen (0.889 vs 0.715). At a cutoff value of 5.591 ng/mL, the diagnostic sensitivity, specificity, and likelihood ratio of serum trefoil factor 3 for colorectal cancer was 74.2%, 94.8%, and 14.25, respectively. Furthermore, the serum trefoil factor 3 levels in early colorectal cancer (TNM stage I, 3.67 ± 1.27 ng/mL) were significantly increased compared to healthy controls (P < .001); however, there was no significant difference compared to patients with polyps (P = .576). We observed that the serum trefoil factor 3 levels decreased after surgery (6.66 ± 2.4 vs 4.48 ± 1.80 ng/mL; P < .001). In addition, high serum trefoil factor 3 levels were associated with poor tumor differentiation and clinical TNM stage (P < .05). In conclusion, serum trefoil factor 3 is a promising biomarker for the diagnosis of colorectal cancer and prognosis of patients.