Extracellular vesicles, more particularly exosomes, are nanostructures of great medical interest. Similarly to other complex biopharmaceuticals such as virus, the purification of these nanovesicles is still a considerable challenge. Here we describe a proof-of-concept purification strategy based on the well-established size exclusion chromatography technique, operated in a semi-continuous mode. A two-column system was designed and experimentally validated. Immunoblotting for protein exosome markers and electron microscopy indicated the presence of exosomes, with its characteristic cup-shaped morphology. The isolated nanovesicles have a mode size of 128 nm and a concentration and size distribution very similar between the collected products. Importantly, after comparison with batch chromatography, we were able to increase the yield up to 83%, specific productivities in 167% and a 2.7-fold reduction in buffer consumption with the semi-continuous operation. Finally, we demonstrate that the developed process can be potentially applied in the large-scale production of exosome-based therapeutics.