Background: Currently, no satisfactory biomarkers are available to screen for esophageal squamous cell carcinoma (ESCC). The goal of this study was to find biomarkers and establish a serum protein fingerprint model for early diagnosis of ESCC using the ClinProt protocol of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods: Serum samples were collected from 62 patients with ESCC, nine patients with esophageal adenocarcinoma (EA) and 38 healthy individuals. Proteomic spectra of mass to charge ratio (m/z) were generated following the application of plasma to weak cationic-exchanger magnetic beads (WCX-MB). The spectral data were analyzed using a support vector machine, and potential biomarkers were chosen for system training and used to construct diagnostic models. Results: Three differential patterns were established using MALDI-TOF MS. Pattern 1, consisting of 11 protein peaks, separated ESCC patients from the healthy individuals with a sensitivity of 90.0% and a specificity of 88.4%. Pattern 2, consisting of eight protein peaks, separated ESCC in stage I and stage II from stage III and stage IV with a sensitivity of 92.9% and a specificity of 82.3%. Pattern 3, consisting of seven protein peaks, separated ESCC from EA with a sensitivity of 91.3% and a specificity of 80.0%. Conclusions: These results suggested that MALDI-TOF MS combined with MB separation yields significantly higher sensitivity and specificity for the detection of serum protein in patients with ESCC. Clin Chem Lab Med 2010;48:855-61.