Objective To explore the different expression of serum proteins of the patients with breast cancer and to screen the diagnostic biomarkers. Methods Tandem mass tag (TMT)-based quantitative proteomics technology was used to detect and quantificationally analyze the serum proteins of 68 stage Ⅱ breast cancer patients and 62 healthy females; the differentially expressed proteins were screened. Bioinformatics analysis of the differentially expressed proteins were performed by UniPort Knowledgebase, Proteome Discoverer software and online software STRING (http://www.string-db.org). The protein and mRNA expressions of screened proteins, vimentin (VIME, up-regulated 3.918 folds than that of healthy females) and Raf-1 threonine-protein kinase (RAF1, down-regulated 0.251 folds) were detected by Western blotting and qPCR, respectively. Results We screened and identified 67 differentially expressed proteins, with 26 significantly up-regulated and 41 significantly down-regulated. Gene ontology (GO) and enrichment analysis showed that the differentially expressed proteins were related to tumor angiogenesis, metabolism and bioadhesive capacity. The protein and mRNA expressions of RAF1 in the patients with stage Ⅱ breast cancer were significantly lower than those in healthy females, and the protein and mRNA expressions of VIME were significantly higher, which was similar to the results of screening analysis. Conclusion TMT-based quantitative proteomics can effectively identify the differentially expressed proteins of stage Ⅱ breast cancer. VIME and RAF1 may be potential serum biomarkers of lymph node metastasis of breast cancer.