Breast cancer is one of the most commonly diagnosed cancers among females worldwide. Early detection of breast cancer is of vital importance to the reduction of the mortality rate. However, the lack of specific biomarkers that can effectively identify breast cancer cells limits the ability for early diagnosis of breast cancer. RNA-cleaving fluorogenic DNAzymes (RFDs), which can be produced through the systematic evolution of ligands by exponential enrichment (SELEX) process, are catalytic DNA molecules capable of generating a fluorescent signal when the appropriate target is bound. In this study, we carried out a SELEX experiment to select for RFDs that are active in the cell lysate of MDA-MB-231, a model breast cancer cell line. We obtained a RFD probe, named AAI2-5, that can detect MDA-MB-231 at a concentration of cell lysate proteins as low as 0.5 μg/mL (which is equivalent to ∼5000 cell/mL). AAI2-5 is capable of distinguishing MDA-MB-231 cells from normal cells as well as other types of tumor cells, including other subtypes of breast cancer cells. Moreover, AAI2-5 responded positively to more than 90% of malignant breast tumors. This report is the first study to explore the RFD system for the detection of cancer cells. The results suggest that RFD can be potentially applied for the diagnosis and treatment of breast cancer in the future.