Background: Peanut allergy is one of the most severe class I food allergies with increasing prevalence. Especially lipophilic allergens, such as oleosins, were found to be associated with severe symptoms, but are usually underrepresented in diagnostic extracts. Therefore, this study focused on isolation, molecular characterization and assessment of the allergenicity of peanut oleosins. Methods and Results: A comprehensive method adapted for the isolation of peanut oil bodies of high purity was developed comprising a stepwise removal of seed storage proteins from oil bodies. Further separation of the oil body constituents, including the allergens Ara h 10, Ara h 11, the presumed allergen oleosin 3 and additional oleosin variants was achieved by a single run on a preparative electrophoresis cell. Protein identification realized by N-terminal sequencing, peptide mass fingerprinting and homology search revealed the presence of oleosins, steroleosins and a caleosin. Immunoblot analysis with sera of peanut-allergic individuals illustrated the IgE-binding capacity of peanut-derived oleosins. Conclusion: Our method is a novel way to isolate all known immunologically distinct peanut oleosins simultaneously. Moreover, we were able to provide evidence for the allergenicity of oleosins and thus identified peanut oleosins as probable candidates for component-resolved allergy diagnosis.