A novel oriented immobilized lipase was derived from Yarrowia lipolytica lipase LIP2 covalently immobilized on functionalized Fe3O4 magnetic nanoparticles (MNPs) in reverse micelles system (RMS). The activity recovery reached 382% compared with 29% in aqueous phase, and further ran up to 1425% under optimum conditions. (3-Aminopropyl) triethoxysilane (APTES) coated Fe3O4 nanoparticles were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). A significant alteration in the secondary structure of the lipase in RMS with a 15.5% increase of alpha-helix content and a 12.5% decrease of beta-sheet content was detected by circular dichroism (CD). The immobilized lipase was employed to enrich polyunsaturated fatty acids in fish oil, a 90% increase of DHA content was obtained after 12 h, and after 20 cycles of successive usage, it still remained over 80% of relative hydrolysis degree, which shows a good recyclability. (C) 2013 Elsevier Ltd. All rights reserved.