Electric fields have been widely used to separate charged DNA molecules in a gel sieving matrix or artificial pillar array within microfluidic devices. It has been shown that the effective electrophoretic (EP) mobility of DNA molecules is dependent on both the size of DNA molecules and the radius of gel matrix or artificial pillar. Large DNA has a decreased EP mobility due to increased collisions with the gel matrix or artificial pillar array. However, the EP mobility of a single DNA molecule in a non-uniform electric field without a gel matrix or artificial pillar is rarely investigated. In this study, electrophoretic DNA dynamics in a straight microchannel and a single microscaled cross-slot were investigated. It was found that the EP mobility of a single DNA molecule is not only dependent on the ion strength of the buffer solution, but also related to the configuration of the DNA molecule and the concentration of surrounding small molecules such as sucrose or glucose. In a zero electric field gradient generated in the straight channel, DNA molecules display a larger value of EP mobility in a high sucrose/glucose concentration solution. While in a constant nonzero electric field gradient produced by single cross-slot, the effective EP mobility of DNA molecule is highly dependent on its aspect ratio. A larger aspect ratio of the DNA molecule leads to a larger effective EP mobility. It is widely accepted that the EP mobility of individual molecules, such as DNA, is constant. However, our findings indicate that the EP mobility of flexible molecules is dependent on the shape of the molecule as it deforms, and the solution it is in. This study provides important fundamentals for the understanding and control of DNA separation with electrophoresis.