As one of the most common cancers, colorectal cancer (CRC) is a major public health issue worldwide. Thus, the identification of novel biomarkers to aid in the early diagnosis of CRC is crucial. The aim of the present study was to identify a novel protein biomarker for CRC, and to identify its structure. In this study, a total of 99 serum samples from 73 CRC patients and 26 healthy controls were collected and analyzed by SELDI-TOF-MS. The biomarkers were separated using HPLC and detected with MALDI-TOF-MS. The qualified peaks were ranked by p-value of non-parametric tests and the top 10 peaks displaying significant differences were selected. Among the 10 protein biomarkers, the concentration of a 3.9‑kDa protein in the serum of the CRC patients was much lower than that in the healthy controls. Therefore, the 3.9‑kDa protein was selected as a biomarker for CRC and its separation and purification were performed. The structure of the 3.9-kDa protein biomarker was determined by LC-MS/MS, and was confirmed to be a fragment of serine/theonine kinase 4 (MST1/STK4). The 3.9‑kDa protein biomarker had high sensitivity and specificity for CRC, and its potential clinical application warrants further investigation.