With the advancement in electrokinetics and its applications to multiphase microfluidic systems, there lacks a fundamental understanding of electroosmotic flow with the presence of immiscible liquid-fluid interfaces. This thesis addresses the fundamentals of two-phase electroosmotic flow by theoretical development and experimentation. A theoretical model is proposed, which considers both the electrical double layer forces and the surface forces at liquid-fluid interfaces. A microfabrication method is also proposed, in which self-assembled monolayers of Octadecyltrichlorosilane and deep-UV irradiation were used to create surface energy (hydrophobic-hydrophilic) patterns simultaneously on all inner surfaces of a slit glass microchip. Liquid is confined by the surface energy on the glass surfaces, forming liquid-air interfaces sidewalls. Experimental observations indicated only the theoretical model that includes the consideration of surface charges is capable of fully describing all of the experimental results.