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Aim: This study aims to explore the presence of informative protein biomarkers in the human serum proteome. Patients and Methods; Serum samples collected from 20 breast cancer patients which were divided into two groups of breast cancer, and 10 health controls and 10 mammary fibroma patients were profiled using iTRAQ technology coupled with LC-ESI-QTOF-MS, and Mascot searching. Western-blotting were used for validation of the candidate biomarkers on a new group of breast cancer and healthy subjects as well as mammary fibroma patients. Results: 335 proteins were identified, and 11 proteins were associated with breast cancer, includind 1 upregulated protein and 10 downregulated proteins. Three biomarker candidates generated from iTRAQ experiments were successfully verified using Western-blotting. Conclusion: This study provided a global view of potential mechanisms and potentional biomarkers of breast cancer, and demonstrated that iTRAQ combined with LC-ESI-QTOF-MS quantitative proteomics is a powerful tool for biomarker discovery.