We report a novel microfluidic system that is capable of rapidly detecting DNA and its mutants in microfluidic droplets, in addition to elucidating the dynamic hybridization process. This microfluidic picoliter droplet analysis system is able to overcome the limitations of conventional analytical techniques that utilize immobilized sensing probes on a substrate. Molecular beacon (MB), a fluorescence resonance energy transfer (FRET) molecule, was used as the DNA sensing probe in picoliter droplets. The MB-DNA duplex formation process was analyzed by the change in FRET signal, which was acquired by the time-resolved method: converting distance traveled to hybridization time. This technique demonstrates the ability to detect presence of target nucleic acids within few seconds, multiplex DNA samples in microdroplet, and distinguish single nucleotide polymorphisms. It is promising for analyzing biomolecules or reactions, such as mRNA, cells, enzymatic activity, and protein folding whose analysis requires rapid mixing and small volume.