The nonuniform motion of charged species in a varying electric field may provide unique separation and focusing power for chemical, biochemical, and nanoscale studies. We imaged in real time the nonuniform motion of single DNA molecules under varying-field isotachophoresis (ITP) conditions. From the trajectories of single molecules, we obtained the time- and position-dependent electric field strength (E) and revealed the behavior of adaption barriers within electro-osmotic flow (EOF)-driven and EOF-independent ITP. We found that the initial terminating electrolyte zone of constant E is split into two zones: a highly adapted high-E zone and a low-E zone of gradually adapting electric field. The formation of the two unique zones is associated with the rate-limiting mass transfer barrier in EOF-driven ITP. As a result of the unique E distribution, DNA molecules first slow to a stop and then rapidly move backward to the leading electrolyte/terminating electrolyte boundary. This provides a novel mechanism for selective focusing of target molecules in dilute solutions of large volume. We show that the ITP focusing can improve the detection of single DNA molecules (limit of detection = 4 × 10(-17) mol/L), which are stochastically distributed at extremely low concentrations. The ITP strategy focuses individual molecules into a small volume that is matched with the focal point of single-molecule imaging.