•Seven oleosin isoforms were identified.•Alkaline extraction could also remove some oleosin isoforms from oil body.•Oleosin isoforms were not glycoproteins and were thiol-containing proteins. Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24kDa; three isoforms, 18kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24kDa oleosin isoforms possessed less thiol groups than 18kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH.