Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 μL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption.