Efficacy of anti-human epidermal growth factor receptor 2 (HER2) treatment is impacted by tissue-based evaluation bias due to tumor heterogeneity and dynamic changes of HER2 in breast cancer. Circulating tumor cell (CTC)-based HER2 phenotyping provides integral and real-time assessment, benefiting accurate HER2 diagnosis. This study developed a semi-quantitative fluorescent evaluation system of HER2 immunostaining on CTCs by peptide-functionalized magnetic nanoparticles (Pep@MNPs) and immunocytochemistry (ICC). 52 newly-diagnosed advanced breast cancer patients were enrolled for blood samples before and/or after first-line treatment, including 24 patients who were diagnosed with HER2+ tumors and treated with anti-HER2 drugs. We enumerated CTCs and assessed levels of HER2 expression on CTCs in 2.0 ml whole blood. Enumerating CTCs at baseline could distinguish cancer patients (sensitivity, 69.2%; specificity, 100%). 80.8% (42/52) of patients had at least one CTCs before therapy. Patients with