The objective of this study was mainly to develop and evaluate a membrane array-based method simultaneously detecting the expression levels of a multiple mRNA marker panel in the peripheral blood for used in complementary breast cancer diagnosis. The mRNA markers employed included cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), c-Met, Her2/neu, and mammaglobin (hMAM). The specimens of peripheral blood were collected from 80 healthy women and 102 female patients with breast cancer. The expression levels of molecular markers were evaluated by real-time Q-PCR and membrane array. Data obtained from real-time Q-PCR and membrane array were subjected to linear regression analysis, revealing that there was a high degree of correlation between the results of these two methods (r=0.979, P<0.0001). The result of membrane array assay with a combined panel of five mRNA markers was demonstrated to achieve sensitivity of 80.6%, and specificity of 83.8% for breast cancer detection, much higher than those of analysis of single marker. In addition, we demonstrated that the membrane array method could detect circulating cancer cells at a density as low as five cancer cells per 1 ml of blood. The analysis of correlation between the outcome of membrane array and clinicopathological characteristics indicated that overexpression of the multiple marker panel was significantly correlated with tumor size (P=0.030) and TNM stage (0.009). In conclusion, the detection of circulating cancer cells by means of membrane array simultaneously monitoring five mRNA markers could significantly enhance the sensitivity and specificity for cancer cell detection.