To analyze the differentially expressed proteins in bronchial epithelial carcinogenesis process and discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC), iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 1036 proteins and 102 differentially expressed proteins were identified. Among these differentially expressed proteins, some proteins are progressively upregulation, some proteins are progressively downregulation in human bronchial epithelial carcinogenic process, and the other proteins are upregulation or downregulation in a certain stage of the process of carcinogenesis. Functional analysis and subcellular localization were performed on 102 differentially expressed proteins. Functional analysis showed that these differentially expressed proteins were associated with metabolic process, apoptosis, cell proliferation, cell differentiation, signal transduction, transcription, translation, cell adhesion, immune response, and development. Western blotting and immunohistochemistry were performed to detect the expression levels of the two proteins (S100A9 and CKB) in NBE, SM, AH, CIS and invasive LSCC, which also support the findings in MS analysis. The results suggest that these differentially expressed proteins associated with bronchial epithelial carcinogenesis, and they may be early diagnostic marker of lung squamous cell carcinoma. We further study the biological function of the differentially expressed proteins, will help to elucidate the bronchial epithelial carcinogenesis mechanism, and provide new ideas for early diagnosis and pathogenesis research of LSCC.