The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fl uidic channels are impor-tant in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties ofλ-DNA molecules transferring along the microchannels driven by the exteal electrickinetic force were systemically investigated with the single molecule fl uorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the exteal electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions: (i) along the direction of the exteal electric field, when the electric field intensity was smaller than a certain threshold value;(ii) opposite to the direction of the exteal electric field, when the electric field intensity was greater than the threshold electric field intensity. The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the infl uence of electro-osmosis fl ow. These new findings will theoretically guide the practical application of fl uidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules.