This paper presents a simple method to enhance the separation efficiency of DNA biosamples in a chip-based capillary electrophoresis system utilizing high-voltage pulsed DC electric fields. Since the average power consumption is reduced by the pulse electric fields, the Joule heating effect is significantly reduced. The experimental and numerical investigations commence by separating a mixed sample comprising two fluoresceins with virtually identical physical properties, namely Rhodamine B and Rhodamine 6G. It is found that the level of separation is approximately 2.1 times higher than that achieved using a conventional DC electric field of the same intensity. The performance of the proposed method is further evaluated by separating a DNA sample of Hae III digested ΦX-174 ladder. Results indicate the separation level of the two neighboring peaks of 5a and 5b in the DNA marker is as high as 1.3 which is extremely difficult to be achieved using a conventional capillary electrophoresis scheme. The improved separation performance of the proposed pulsed DC electric field approach is attributed to a lower Joule heating effect as a result of a lower average power input and the opportunity for heat dissipation during the zero-voltage stage of the pulse cycle. CE separation using highfrequency DC pulse electric fields can be an even simple and efficient way to control the Joule heat in the separation channel during separation.