Extracellular vesicles (EVs) have emerged as vehicles that mediate diverse cell-cell communication. However, in-depth understanding of these vesicles is hampered by a lack of a reliable isolation method to separate different types of EVs with high levels of integrity and purity. Here, we developed a nanoporous and ultra-thin membrane structure (NUTS) that warrants the size-based isolation of EVs without cake formation, minimizing the sample loss during the filtration process. By utilizing the micro-electro-mechanical systems (MEMS) technique, we could also control the pore size in nanoscale. We validated the performance of this membrane to separate EVs according to their size range.