Plasma is a complex substance that contains proteins and circulating nucleic acids and viruses that can be utilised for clinical diagnostics, albeit a precise analysis depends on the plasma being totally free of cells. We proposed the use of a dielectrophoresis (DEP)-active hydrophoretic method to isolate plasma from blood in a high-throughput manner. This microfluidic device consists of anisotropic microstructures embedded on the top of the channel which generate lateral pressure gradients while interdigitised electrodes lay on the bottom of the channel which can push particles or cells into a higher level using a negative DEP force. Large and small particles or cells (3 μm and 10 μm particles, and red blood cells, white blood cells, and platelets) can be focused at the same time in our DEP-active hydrophoretic device at an appropriate flow rate and applied voltage. Based on this principle, all the blood cells were filtrated from whole blood and then the plasma was extracted with a purity of 94.2% and a yield of 16.5% at a flow rate of 10 μL min−1. This solved the challenging problem caused by the relatively low throughput of the DEP based device. Our DEP-active hydrophoretic device is a flexible and tunable system that can control the lateral positions of particles by modulating the external voltages without redesigning and fabricating a new channel, and because it is easy to operate, it is easily compatible with other microfluidic platforms that are used for further detection.