Rapid molecular diagnosis using nucleic acid biomarkers is important for timely identification of acute pathogenic infections. We introduce an active kinetic approach called sensing single molecule under micromanipulation (SSM3) to quantify nucleic acids, which circumvents hydrodynamic limits of reaction rate, sensitivity and specificity. We demonstrate the 15-minute assay to detect synthetic miRNAs with subfemtomolar limit of detection and high-confidence discrimination of single-base-pair mismatch.