Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. The lower survival rate of ESCC is attributed to late diagnosis and poor therapeutic efficacy; therefore, the identification of tumor-associated proteins as biomarkers for early diagnosis, and the discovery of novel targets for therapeutic intervention, seems very important for increasing the survival rate of ESCC. To identify tumor-associated proteins as biomarkers in ESCC, we have analyzed ESCC tissues and adjacent normal tissues by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The results showed that a total of 104 protein spots with different expression levels were found on 2DE, and 47 proteins were eventually identified by MALDI-TOF MS. Among these identified proteins, 33 proteins including keratin 17 (KRT17), biliverdin reductase B (BLVRB), proteasome activator subunit 1 (PSME1), manganese superoxide dismutase (MnSOD), high-mobility group box-1(HMGB1), heat shock protein 70 (HSP70), peroxiredoxin (PRDX1), keratin 13 (KRT13), and so on were overexpressed, and 14 proteins including cystatin B (CSTB), tropomyosin 2 (TPM2), annexin 1 (ANX1), transgelin (TAGLN), keratin 19 (KRT19), stratifin (SFN), and so on were down-expressed in ESCC. Biological functions of these proteins are associated with cell proliferation, cell motility, protein folding, oxidative stress, and signal transduction. In the subsequent study using immunoassay on ESCC serum samples and tissue-array slides, two representative proteins, HSP70 and HMGB1, were selected as examples for the purpose of validation. The results showed that both HSP70 and HMGB1 can induce autoantibody response in ESCC sera and have higher expression in ESCC tissues. Especially, the frequency of antibodies to HSP70 in ESCC sera was significantly higher than that in normal human sera. The preliminary results suggest that some of these identified proteins might contribute to esophageal cell differentiation and carcinogenesis, certain proteins could be used as tumor-associated antigen (TAA) biomarkers in cancer diagnosis, and further studies on these identified proteins should provide more evidence of how these proteins are involved in carcinogenesis of ESCC.