An effective enrichment and identification method of Circulating Tumor Cells (CTC) was clinically validated and the specificity and sensitivity of the method in CTCs detection from breast cancer patients was evaluated as well in this paper. Fluorescent labeled cultured breast cancer cell line, SKBR-3 cells were added into 7.5mL peripheral blood obtained from ten of healthy volunteers respectively in this paper. After enrichment by using anti-leukocyte antigen antibody conjugated nanobeads, the slides were observed with fluorescent microscope and the fluorescent labeled cells were counted. The enrichment efficiency was evaluated by average cells recovery rate. The peripheral bloods from 20 of healthy volunteers and 15 of patients with clinical diagnosis of invasive breast cancer were extracted and blind coded. Then the CTCs were enriched and identified by immunofluorescent plus immunocytochemical stains. By comparing the difference of CTCs number between healthy volunteers and breast cancer patients, the specificity and sensitivity of the method were evaluated. The results show that the average spiked cells recovery rate was 75%. The spiked cells were anti-keratin (CK18) stain positive, anti-Her2 stain positive and anti-CD45 stain negative, which consist with the antigen expression characters of breast cancer cells. The unblinding result revealed that there were none of CTCs found in bloods from 20 of healthy volunteers, while among 15 of breast cancer patients, less than 3 CTCs (0—2) were detected in 33.3% patients and more than 3 CTCs were detected in 66.7% patients. The enrichment and identification method used could well recover spiked cells in peripheral blood and have significant specificity and sensitivity in CTCs clinical detection.