Breast cancer has become one of the most common malignant tumors among women, and axillary lymph node (ALN) metastasis is the most important mode of breast cancer metastasis, which is the most important indicator to estimate the prognosis and guide the decision of postoperative adjuvant treatment. Axillary lymph node dissection (ALND) is the most accurate way to evaluate ALN status, and meanwhile is the main cause of complications such as upper extremity lymphedema, pain, and sensory impairment. With the increased detection of early-stage breast cancer, ALN negative breast cancer has accounted for more than half of all breast cancer patients, if ALND is performed in all breast cancer patients, only a small number of patients will benefit from it, and the majority of patients are overtreated. Sentinel lymph nodes (SLNs) are the first barrier of lymph node metastasis in patients with breast cancer. After decades of development, sentinel lymph node biopsy (SLNB) has become a standard procedure for ALN staging in breast cancer patients and is often used to determine the choice of treatment. Accurate and rapid intraoperative diagnosis of SLN enables SLN-negative breast cancer patients to avoid ALND, and SLN-positive patients to complete ALND through a single operation to avoid the cost burden and surgical risk of a second operation. Frozen section and touch imprint cytology examinations are commonly used to detect intraoperative metastatic SLN. These two routine pathological tests only detect representative sections of SLN. Both of them have shortcomings such as low sensitivity, subjectivity, non-standardization, small amount of detected tissue (far less than 5%), and no unified diagnostic criteria. Therefore, a new detection method with accurate results and simple operation is urgently needed in clinical practice. In recent years, molecular diagnostic techniques have developed rapidly. One-step nucleic acid amplification (OSNA) is a molecular diagnostic method for accurate detection of intraoperative lymph node metastasis of breast cancer through reverse transcriptional loop mediated isothermal amplification of targeted cytokeratin 19 mRNA. The operation time of OSNA detection is about 30 to 40 min. In SLN detection and analysis, the accuracy and sensitivity of OSNA are better than those of the conventional pathological detection methods. Rapid intraoperative quantification can distinguish the macrometastases from micrometastases and guide surgical protocols. In addition, OSNA is superior in detecting SLN micrometastases. Here, the authors address the progress and application prospect of OSNA detection in SLN metastasis of breast cancer.