Real time direct observation of single molecule using fluorescent technique has elucidated various biological phenomena, because not only average but also distributions and fluctuations of molecules can be characterized directly by this method, on the other hand, conventional methods can observe only average behaviors of molecules. In recent years, single-molecule analyses of various DNA-protein interactions by using florescent techniques were performed. In this study, single-molecule DNA hydrolysis by exonucleaseIII was observed, which has 3′ → 5′ exonuclease activity. This single-molecule observation method was based on optical trap and two-layer laminar flow, in which mixing between the two layers was negligible. First of all, one DNA-bead complex was optically trapped in a layer without exonucleaseIII. This trapping permitted stretching of DNA by flow. ExonucleaseIII reaction was initiated by moving the trapped DNA-bead complex to another flow layer containing exonucleaseIII, then shortening of fluorescently stained DNA was observed in real time. The sequentially captured photographs demonstrate that the digested DNA molecule linearly shortened with reaction time. The digestion rate from single-molecule experiment was determined and compared to in vitro experiment. As a result, digestion rate of exonucleaseIII from single-molecule experiment was ∼20 times higher than in vitro digestion rate.