Vortex-based separation is a promising method in particle–particle separation and has only been demonstrated theoretically some years ago. To date, a continuous-flow separation device based on vortices has not been conceived because many known vortices were either unstable or controlling them lacked precision. Electro-convection from induced charge electro-osmosis (ICEO) has advantages, such as adjustable flow profiles, long-range actuation, and long-lived vortices, and offers an alternative means of particle separation. We found though a different ICEO focusing behaviour of particles whereby particles were trapped and concentrated in two vortex cores. Encouraged by these features of ICEO vortices, we proposed a direct method for particle separation in continuous flow. In various experiments, we first characterized the ICEO-induced focusing performances of various kinds of particle samples in a straight channel embedded with an individual central bipolar electrode, presenting a justifiable explanation. Second, the combined dependences of ICEO particle separation on the sample size and mass density were investigated. Third, an application to cell purification was performed in which we obtained a purity surpassing 98%. Finally, we investigated the ICEO characteristics of nanoparticles, exploiting our method in isolating nanoscale objects by separating 500 nm and 5 μm polystyrene beads, gaining clear separation. Certain features of this method, such as having ease of operation, simple structure, and continuous flow, and being prefocusing free and physical property-based, indicate its good potential in tackling environmental monitoring, cell sorting, chemical analysis, isolation of uniform-sized graphene and transesterification of micro-algal lipids to biodiesel.