This work presents a novel electrophoretic microchip design which is capable of directly coupling with flow-through analyzers for uninterrupted sampling. In this device, a 3 mm wide sampling channel (SC) was etched on quartz substrate to create the sample inlet and outlet and the 75 microm wide electrophoretic channels were also fabricated on the same substrate. Pressure was used to drive the sample flow through the external tube into the SC and the flow was then split into outlet and electrophoretic channels. A gating voltage was applied to the electrophoretic channel to control the sample loading for subsequent separations and inhibit the sample leakage. The minimum gating voltage required to inhibit the sample leakage depended on the solution buffer and increased with the hydrodynamic flow-rate. A fluorescent dye mixture containing Rhodamine B and Cy3 was introduced into the sample stream at either a continuous or discrete mode via an on-line injection valve and then separated and detected on the microchip using laser-induced fluorescence. For both modes, the relative standard deviation of migration time and peak intensity for consecutive injections was determined to be below 0.6 and 8%, respectively. Because the SC was kept floating, the external sampling equipment requires no electric connection. Therefore, such an electrophoresis-based microchip can be directly coupled with any pressure-driven flow analyzers without hardware modifications. To our best knowledge, this is something currently impossible for reported electrophoretic microchip designs.