Micro- and nanopillar chips are widely used to separate and enrich biomolecules, such as DNA, RNA, protein, and cells, as an analytical technique and to provide a confined nanospace for polymer science analyses. Herein, we demonstrated a continuous accurate and precise separation technique for extracellular vesicles (EVs), nanometer-sized vesicles (typically 50-200 nm) currently recognized as novel biomarkers present in biofluids, based on the principle of electroosmotic flow-driven deterministic lateral displacement in micro- and nanopillar array chips. Notably, the easy-to-operate flow control afforded by electroosmotic flow allowed nanoparticles 50-500 nm in size, including EVs, to be precisely separated and enriched in a continuous manner. By observation of the flow behavior of nanoparticles, we found that electroosmotic flow velocity in the nanopillar arrays did not solely depend on counterion mobility on the surface of nanopillar chips, but rather showed a parabolic flow profile. This hydrodynamic pressure-free and easy-to-use separation and enrichment technique, which requires only electrode insertion into the reservoirs and electric field application, may thus serve as a promising technique for future precise and accurate EV analysis, reflecting both size and composition for research and potential clinical diagnostic applications.