Effects of concentration gradient on double-stranded DNA (dsDNA) separation by capillary electrophoresis are presented. By using a concentration gradient in the range between 0.8% and 3.2% for poly(N,N-dimethylacrylamide) (PDMA), the presence of a mesh-size gradient in the capillary could enhance the separation of larger size DNA fragments, better than that based on a single uniform concentration over the same capillary length. A decrease in the column length could make the gradient effect more obvious. An optimal capillary length could be achieved by using a judicious combination of the concentration gradient and the concentration range, yielding a maximum resolution for the system. The standard deviation of the migration time measured for each DNA fragment was less than 5% in ten continuous runs, suggesting that the gradient formed inside the column was quite stable.