Particle separation is a crucial step in sample preparation processes. The preparation of low volume samples is especially important for clinical diagnosis and chemical analysis. The advantages of microfluidic techniques have lead them to become potential candidates for particle separation. However, existing microfluidic devices require external pumping sources and extensive geometric patterns to attain high separation efficiency, which is disadvantageous when handling low volume samples. This paper presents a low sample volume particle separation microfluidic device with low voltage electrokinetic pumping based on circular travelling-wave electroosmosis (TWEO). Computational numerical software was utilized to simulate two electrokinetic mechanisms: circular TWEO and dielectrophoresis (DEP). The circular TWEO shear flow generates a velocity gradient in the radial direction which causes a shear stress-induced force to drag particles into the center region of the device. In contrast, the non-parallel electrodes induce negative DEP forces which push polystyrene beads towards the peripheral regions; the magnitude of the DEP forces are dependent on the sizes of the polystyrene beads. We used particles of various sizes to experimentally prove the concept of particle separation. Our experiments show that 15 μm beads are dragged into the center region due to the shear stress-induced force, and 1 μm beads move towards the outer region because of the large negative DEP force. The results show a separation purity of 94.4% and 80.0% for 15 μm and 1 μm beads respectively. We further demonstrated particle isolation from a sample of containing a small proportion of 6 μm beads mixed with 1 μm beads at a concentration ratio of 1 : 300. Therefore, the innovative device developed in this paper provides a promising solution to allow particle separation in sample volumes as low as 50 nL.