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Ferrofluids are stable dispersions of nano-sized magnetic particles coated by organic surfactants to prevent them from aggregation. For advanced medical applications, surfactants are chosen based on their ability to form complex bonding with immunoassays. We report on structural investigations of two identical Magnetite-ferrofluids with oleoylsarkosine as surfactant, which are stabilized in different solutions in water or in toluene. Combining contrast variation using polarized neutrons (SANSPOL) in combination with H/D isotope variation of solvents allows different magnetic and non-magnetic structural units of ferrofluids to be identified by small angle neutron scattering. Three structural parts, core-shell particles, aggregates and excess surfactants were found in both ferrofluids. Their size distributions and volume fractions were derived. As a main result the shell thickness of the surfactant, oleoylsarcosine, is found to be clearly different in water (2.3 nm ± 0.1 nm) and toluene (1.9 nm ± 0.1 nm) based ferrofluids. From that we conclude to have a bilayer formation in water and a monolayer in toluene. This indicates that bonding of the surfactants to the magnetic core is mediated via the hydrophilic acid group.