The early detection of breast cancer, the most common malignant tumor disease in women worldwide, relies on mammography and self breast examination. Here we evaluated the concentration of nicotinamide phosphoribosyltransferase (NAMPT), vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor-2 (HER2) in serum and their expression in breast tissues associated with the clinicopathological features of patients with benign and malignant breast tumors. The immunohistochemical analysis showed that NAMPT, VEGF and HER2 proteins were overexpressed in breast tumors. The highest expression was observed in malignant tumors, low in benign tumors and negative in the adjacent normal tissue, indicating that the triplets may be progression markers and correlated with each other. The detection rate of NAMPT, VEGF and HER2 alone in tissue was 54.17, 64.58 and 60.42%, respectively, and was increased to about 79% in double combination and to 90% in triple combination. The basal levels of serum NAMPT, VEGF and HER2 in healthy controls were 94.90 +/- 4.24 pg/ml, 87.0 +/- 2.41 pg/ml and 1.12 +/- 0.04 ng/ml, respectively, measured by ELISA and found to be increased by 6.64-, 1.76- and 2.52-fold, respectively, in patients with malignant breast tumor. These elevated serum levels of NAMPT,VEGF and HER2 in patients were decreased after tumor removal, suggesting that these molecules are the indicators of treatment efficacy. The combined measurement of these triplets together may improve the sensitivity of breast cancer diagnosis and may potentially be used as a testing panel for the detection of malignant tumors, the assessment of treatment effectiveness and the monitoring of the disease progression in patients with breast cancer. Thus, we propose that the biomarker triplet NAMPTNEGF/HER2 can be used as a de novo detection panel for the diagnosis and prognosis of human breast cancer.