The ability to measure many single molecules simultaneously in larger and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM3) to circumvent the above limits. In SSM3, the transportation rate of analyte molecules and the kinetics of molecular interaction are fine-tuned by the nanoparticle micromanipulation. The heterogeneous lifetime of molecular complexes is quantified to discriminate specific binding from nonspecific background noise. By the highly-specific digital counting of single molecules, we demonstrate 15-minute assays for direct detection of microRNAs and amyloid-β proteins via electrical or magnetic micromanipulation, with the limit of detection at the subfemtomolar level. The presented approach could inspire more practical applications of single molecule sensors.