HER2 analysis in circulating tumor cells (CTCs) may have clinical significance for HER2-targeted therapy as HER2-positive CTCs and disseminated tumor cells can be detected in patients with HER2-negative primary tumors who currently do not have access to HER2-targeted therapy. In this study, we performed quantitative analysis by confocal microscopy assay for evaluation of HER2 expression in individual tumor cells. HER2 testing by confocal microscopy assay exhibited high concordance with results of real-time PCR, Western blot analysis and FISH analysis. We found that there was a significant positive correlation between HER2 overexpression and gene amplification in individual CTCs, which provided validation of confocal microscopy assay for HER2 expression in CTCs. By using subsets of 10 consecutive cells (bins), we conclude that HER2 expression (3+) in CTCs predicts HER2 overexpression of tumor with high probability in breast cancer patients. These results may provide interpretive guidelines for HER2 status assay in CTCs and raise great opportunities for using CTCs as non-invasive and 'real-time' biopsy to examine and monitor the status of tumor markers.