The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.