Most previous studies of circulating tumor cells (CTCs) are based on the CellSearch platform, but CellSearch has a number of limitations. This study aimed to use the LiquidBiopsy system and immunofluorescence to test the human epidermal growth factor receptor 2 (HER2) status of CTCs in patients with breast cancer. The LiquidBiopsy system was used to detect HER2-positive (HER2+) cells in whole blood by microfluidic immunomagnetic bead screening and immunofluorescence assay, according to the manufacturer;s instructions. HER2 expression on CTCs was assessed using the Ariol system, calibrated through spiking experiments of 100 cells (BT474, SKBR3, A431, and MDA-MB-231) and 2.5 × 107 white blood cells/mL from healthy donors. Seventy-one patients with breast cancer and 107 non-cancer donors consented to provide blood. Based on breast cancer cell lines experiments, HER2+ CTCs were defined as CTCs with HER2 immunofluorescence intensity ≥ 3.5 times higher than the CD45 immunofluorescence intensity (100% sensitivity and 99.9% specificity). Among the 71 patients with breast cancer, 31 (43.7%) had HER2+ tumor. Among the HER2+ patients, 41.9% (13/31) were found to be HER2+ based on CTC ≥ 1, and 25.8% (8/31) were positive based on CTC ≥ 3. In HER2-negative patients by pathologic examination, 1 (2.5%) patient was found to have ≥ 3 HER2+ CTCs, whereas 15 (37.5%) patients had ≥ 1 HER2+ CTC. HER2+ CTCs were detected at all stages, even in early breast cancer, but the detection rate was higher in metastatic breast cancer. This proof-of-concept study strongly suggests that HER2+ CTCs can be detected using the LiquidBiopsy system. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.