A novel microchip separation system for microparticles based on electromagnetophoresis (EMP) was developed. In this system, focusing and separation of flowing microparticles in a microchannel could be performed by staggered-EMP by controlling the electric current applied to the channel locally combined with the split-flow system for fractionation of eluates. To apply the electric current through the flushing medium in the microchannel, a hollow fiber-embedded microchip with multiple electrodes was fabricated. The hollow fiber was made by a semi-permeable membrane and could separate small molecules. This microchip allowed us to apply the electric current to a part of the microchannel without any pressure control device because a main channel contacted with the subchannels that had electrodes through the semi-permeable membrane. Moreover, the separation using this microchip was combined with the split-flow system at two outlets to improve separation efficiency. Using this system, with the split-flow ratio of 10:1, 87% of 3 μm polystyrene (PS) latex particles were isolated from a mixture of 3 and 10 μm particles. Even the separation of 6 and 10 μm PS particles was achieved with about 77% recovery and 100% purity. In addition, by controlling the applied current, size fractionation of polypropylene (PP) particles was demonstrated. Moreover, biological particles such as pollens could be separated with high separation efficiency by this technique.