Background: 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 family 3 (TFF3) and evaluate the diagnostic accuracy of TFF3 in patients with colorectal cancer. Methods: We prospectively collected serum samples from 72 subjects (36 patients with colorectal cancer and 36 healthy controls) in the derivation cohort. The validation cohort prospectively included 70 subjects (35 patients with colorectal cancer and 35 healthy controls). The levels of serum TFF3 and carcinoembryonic antigen (CEA) were examined using enzyme-linked immunosorbent assay. Results: The serum levels of TFF3 in patients with colorectal cancer was significantly higher compared with that in the healthy controls (9.34±12.2 ng/mL vs. 3.60±1.58 ng/mL, P = 0.008) in the derivation cohort. Using a cutoff of 3.85 ng/mL in the derivation cohort, the sensitivity, specificity, and area under the curve of serum TFF3 for the detection of advanced colorectal cancer were 0.972, 0.611, and 0.875, respectively. The sensitivity of serum TFF3 for the detection of colorectal cancer was superior (97.2%) to that of CEA (36.1%) in the derivation cohort. In the validation cohort, the sensitivity, specificity, and area under the curve of serum TFF3 for the detection of colorectal cancer were 0.771, 0.486, and 0.751, respectively. Similarly, the sensitivity of serum TFF3 for the detection of colorectal cancer was also superior (77.1%) to that of CEA (22.9%) in the validation cohort. Conclusions: Serum TFF3 is a potential biomarker for the detection of colorectal cancer and is more effective compared with CEA.