We demonstrate that electrophoresis on a flat Si substrate is an effective method in separation of DNA with different configurations, e.g., linear, supercoiled, and relaxed or DNA of different length, e.g., supercoiled DNA ladder. The surface separation arises from the different number of contacts due to the conformational differences between adsorbed DNA chains. Imposing a Au nanopattern on the Si surface further improves the separation effect. The simulation of electric field on this patterned surface by the finite element method shows that Au nanodots act as local pinning points for DNA segments due to dielectrophoretic force. The results of molecular dynamics simulation showed that the conformational differences between adsorbed polymer chains were amplified on the patterned surface and enhanced separations were achieved, which are consistent with the experimental results.