Circulating microRNAs have emerged as potential diagnostic biomarkers. The deregulation of the microRNA miR-99a-5p has been previously described as an effective biomarker of early breast cancer. Herein, we present a new nanoporous anodic alumina (NAA)-based biosensor that can detect plasma miR-99a-5p with high sensitivity and selectivity. NAA pores are loaded with rhodamine B and capped with a specific oligonucleotide that is able to block cargo release until the target is present. In the presence of miR-99a-5p, the capping oligonucleotide recognizes the miR-99a-5p sequence and displaces it allowing the release of the encapsulated dye. This method is able to successfully distinguish healthy controls from breast cancer patients, even at early stages with high efficiency, showing the presented system as a promising tool for breast cancer detection.