Due to the lack of portable, field-deployable diagnostic tests for detecting cancer cells, especially for breast cancer, current detection techniques involve the collection of blood and/or tissue samples, which then need to be sent to laboratories for further analysis before decisions can be made by the treating physicians. Conventional techniques such as mammograms and blood analysis are time consuming, requiring specialized technical personnel, in addition to large and expensive laboratory equipment. Herein, we report the development of a novel sensing method to detect breast cancer-specific microRNAs (miRNAs) captured using a complementary sequence binding technique, and we quantify the mechanism using time-resolved Forster resonance energy transfer (TR-FRET). Using terbium-cryptate, the proposed technique reduces the number of steps required to detect the analyte biomarker in clinical serum samples. We also demonstrate the dual detection of biomarkers using the DNA supporter sequence in the proposed TR-FRET technique. We provide a validated proof-of-concept for a minimally invasive, breast cancer-specific, biomarker detection assay, and demonstrate detection limits in the picomolar range using microfluidics as a detection platform for clinical serum samples. The developed microfluidic biosensor has the potential for use as a portable, field-deployable, and highly sensitive diagnostic tool for the rapid and early detection of breast cancer-specific miRNA signatures.