Surface roughness is present in most microfluidic devices as a result of the microfabrication techniques or particle adhesion. It is highly desirable to understand the roughness effect on microscale transport processes. In this study, we developed a 3-D, finite-volume-based numerical model to simulate electroosmotic transport in microchannels with rectangular prism rough elements on the surfaces. Various configurations of roughness were investigated, and the results show different degrees of an even-out effect on liquid transport due to the roughness-induced local pressure field and the variation of the electroosmotic slip boundary velocities. 3D-sample transport through rough microchannels was analyzed. The results demonstrate that the sample's transport under the electrical field is much faster in the pathway between the rough elements; the concentration field in the height and width direction is not uniform. The influence of the electrokinetic properties on liquid flow and sample transport was studied. It was found that the increase of the electroosmotic mobility or the decrease of the electrophoretic mobility can dramatically enhance the uniformity of the concentration field.