This research presents a detection scheme for analyzing the temperature distribution produced by the Joule heating effect nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system¡Xtotal internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the inner channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective-type TIRFM, microscope cover glass are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM makes the intensity information obtained is not sensitive to the channel depth variation which ususally biases the measured results while using conventional epi-fluorescence microscope (Epi-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within hundreds of nanometer of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. In order to investigate the temperature distribution produced by the Joule heating effect for electrokinetically driven microchips, this study not only measures the temperature on the microchannel wall by the proposed TIRFM but also measures the temperature inside the microchannel by an Epi-FM. In addition, this research presents a method to reduce the Joule heating effect and enhance the separation efficiency of DNA biosamples in a chip-based capillary electrophoresis (CE) system utilizing pulse DC electric fields. Since the average power consumption is reduced by the pulse electric fields, the Joule heating effect can be significantly reduced. Results indicate the proposed TIRFM method provides higher measurement sensitivity over the Epi-FM method. Significant temperature difference along the channel depth measured by TIRFM and Epi-FM is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the Joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a simulation model for precisely predicting the Joule heating effect in microfluidic chips. Moreover, in the research of reducing the Joule heating effect and enhancing the separation efficiency in a chip-based CE system utilizing pulse electric fields, the experimental and numerical investigations commence by separating a mixed sample comprising two fluoresceins with virtually identical physical properties. The separation level is approximately 2.1 times higher than that achieved using a conventional DC electric field. The performance of the proposed method is further evaluated by separating a DNA sample of Hae III digested £XX¡V174 ladder. Results indicate the separation level of the two neighboring peaks of 5a (271 bp) and 5b (281 bp) in the DNA ladder is as high as 120% which is difficult to be achieved using a conventional CE scheme. The improved separation performance 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. Overall, the results demonstrate a simple and low-cost technique for achieving a high separation performance in CE microchips.