Oleosins are unique proteins that are crucial for the stabilization of the oil droplets, that nature designed to store and protect triacylgycerols in seeds. To better understand and possibly replicate the role of oleosins in the stability of oil droplets, an efficient extraction for oleosins is necessary, which has not been achieved yet. Oleosins consist of a long central hydrophobic hairpin that is attached to two hydrophilic arms. This high amphiphilicity makes their extraction a challenging task. The aim of the present work was to develop a scalable method to extract oleosins from rapeseed oleosomes using washing steps with methanol, hexane, and ethanol (MHE). Following this method, we obtained oleosins with a recovery of 94 ± 1.4 wt% and a purity of 87.1 ± 1.9 wt%. The recovery was significantly higher (p < 0.005) compared to the commonly applied Folch extraction (recovery of 57.2 ± 5.5 wt%). Oleosins formed micro- and nanosized aggregates when dispersed in aqueous solutions, because of their long hydrophobic moiety. The fraction of nanosized aggregates was 6-fold higher for the oleosins obtained with the MHE method in comparison to those obtained using the Folch method. Due to the smaller aggregates, oleosins obtained using the MHE method were more efficient in reducing the oil-water interfacial pressure and formed a stronger interfacial film in comparison to those obtained with the Folch method. The highly efficient and scalable oleosin extraction, paves the way for elucidating the stabilizing role of oleosins and the way towards industrial oleosin extraction.