HER2 detection is important for breast cancer (BC) treatment and prognosis, but the detection methods currently used have some disadvantages. Quantum dots (QDs)-based probes provide a potentially important new method for HER2 detection in clinical practice. This potential is examined in this paper. A QDs HER2 probe kit and QDs image acquisition and analysis software were developed and applied to 94 clinical samples of BC. Compared to conventional immunohistochemistry techniques, this method provided a superior accurate and sensitive method for the detection of HER2 in clinical breast cancer diagnosis.