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Simple Summary A diagnostic biomarker for the detection of breast cancer remains an unmet clinical need despite decades of intensive research efforts. Herein, we describe, for the first time, the use of nuclease activity as a biomarker to discriminate between healthy and cancer biopsy samples. We have identified a panel of three nucleic acid probes able to target nucleases derived from breast cancer tumors with high sensitivity and specificity. These results are in good agreement with histopathological analysis as the diagnostic gold standard. Moreover, these findings support nuclease activity as a potential adjacent diagnostic tool and shed light on the use of nuclease activity as a detection biomarker in breast cancer. Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic. Funding Agencies|Wallenberg Centre for Molecular Medicine (WCMM) Linkoping, Sweden; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University (Faculty Grant SFO-Mat-LiU) [2009-00971]; program Torres Quevedo (MINECO); industrial doctorate program (MINECO)