Objective: Biomarker assay is a noninvasive method for the early detection of esophageal squamous cellcarcinoma (ESCC). Searching for new biomarkers with high specificity and sensitivity is very important for the earlydetection of ESCC. Serum surface-enhanced laser desorption/ionization-time of flight mass spectrometry(SELDI-TOF-MS) is a high throughput technology for identifying cancer biomarkers using drops of sera. Methods: Inthis study, 185 serum samples were taken from ESCC patients in a high incidence area and screened by SELDI. Asupport vector machine (SVM) algorithm was adopted to analyze the samples. Results: The SVM patterns success-fully distinguished ESCC from pre-cancerous lesions (PCLs). Also, types of PCL, including dysplasia (DYS) and basalcell hyperplasia (BCH), and healthy controls (HC) were distinguished with an accuracy of 95.2% (DYS), 96.6% (BCH),and 93.8% (HC), respectively. A marker of 25.1 kDa was identified in the ESCC patterns whose peak intensity wasobserved to increase significantly during the development of esophageal carcinogenesis, and to decrease obviously after surgery. Conclusions: We selected five ESCC biomarkers to form a diagnostic pattern which can discriminateamong the different stages of esophageal carcinogenesis. This pattern can significantly improve the detection ofESCC.