Manipulating a single DNA molecule and effectively introducing it into and exporting micro-nano-fluidic channels are prerequisites for the functional DNA biochips. And it is the key to the precise separation and screening of different DNA molecules by the micro-/nanochannel system that accurately understanding the movement characteristics and dynamic mechanism of DNA molecules moving near the channel port. In this paper, the electrodynamic characteristics of λ-DNA molecule entering into/leaving off a 50 μm channel port driven by the electric field force are systematically investigated and analyzed by the single molecule fluorescence microscopy. The experimental results indicated that there were the maximum (E_max) and minimum (E_min) thresholds of the applied electric field intensity, and only when the field intensity E meets E_min ≤ E ≤ E_max, the single λ-DNA molecule could successfully enter into the trans port and exit out of the cis port; when the electric field intensity was less than the minimum threshold, E ≤ E_min, λ-DNA molecules could not enter the trans port; when the electric field intensity was greater than the maximum threshold, E_max ≤ E, λ-DNA molecules could move into the microchannel through the trans port, but not exit out of the cis port. When λ-DNA molecule migrated toward the cis port along the channel, the movement state was changed, some new phenomena were observed, e.g. the translocation direction was reversed, reciprocated, or even rotated; moreover, the DNA molecules were easy to adhere to the channel wall. In addition, when the electric field intensity enhanced, the distance between the position where DNA molecular direction reversing and the cis port was increased. Based on the microfluidic electrodynamics, the physical mechanism of the velocities and translocation states of single λ-DNA molecule passing microchannel port was preliminarily analyzed. The results of this study have certain practical guiding significance for the development of gene chip laboratory and DNA molecular sensors based on the micro/nanochannel fluidic system.