Esophageal squamous cell carcinoma (ESCC) is a common digestive tract malignant tumor with high incidence and dismal prognosis throughout the world. However, the reliable biomarkers for clinical diagnosis and the comprehensive protein signaling regulatory networks for ESCC remain to be further elucidated. The serum proteome may provide valuable clues for early diagnosis of ESCC and discovery of novel molecular insights. In the current study, an optimized proteomics approach was employed to discover novel serum-based biomarker panel for ESCC, as well as unveil abnormal signal pathways. Gene ontology (GO) enrichment analysis was done by Gene Set Enrichment Analysis (GSEA) or Metascape database, respectively. Pathway analysis was accomplished by GeneCards database. The correlation coefficient was assessed using Pearson and distance correlation analyses. Prioritized candidates were further verified in two independent validation sets by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) staining. A total of 633 non-redundant proteins were identified in serum of patients with ESCC, of which 59 proteins displayed more than 1.5-fold increase, whereas 10 ones showed 1.5-fold decrease compared with healthy controls. Verification was performed for candidate biomarkers including ENO1, TPI1, PGAM1, S100A8/A9 and SAA1. Receiver operating characteristics (ROC) curve showed a biomarker panel composed of three protein molecules including S100A8/A9 and SAA1 with a high diagnostic sensitivity and specificity, the area under characteristic curve (AUC) is up to 91.4%. Differentially expressed proteins were subjected to functional enrichment analysis which uncovered the dysregulation of signaling pathways mainly involving in glycolysis, TLR4, HIF-1, Cori cycle, TCA cycle, folate metabolism, and platelet degranulation. More importantly, activated glycolysis and TLR4 pathways were strongly positively correlated with unfavorable clinicopathological TNM stages in ESCC.