Magnetite nanoparticles have unique properties including superparamagnetism and low toxicity. They have been used as supports for immobilization of enzymes because of the advantage including easy separation and effective recycle under external magnetic field. Therefore, the present study aimed at developing a new enzymatic biocatalyst from the immobilization of Yarrowia lipolytica’s (IMUFRJ 50682) lipase on magnetic nanoparticles for its use in free fatty acids and carotenoids extraction from palm oil (Elaeis guineenses). The lipase production conducted in a 4 L benchtop bioreactor generated a crude enzyme extract with hydrolytic p-nitrophenyl laurate activity of 58 U/ml after 24 h. Lipase was immobilized by adsorption on magnetite nanoparticles (Fe3O4). The crude enzymatic extract, both free and immobilized, were used in the hydrolysis of palm oil. Temperature, reaction time and substrate ratio (water/palm oil) were evaluated in a central composite experimental design. The initial concentration of fatty acids and carotenoids present in palm oil obtained via traditional extraction (using organic solvents) were 82.45% and 1892.3 mg/kg, respectively. Using crude lipase extract immobilized in nanoparticles, after 120 min, temperature of 24°C and water/oil ratio of 2, it was possible to obtain similar values, with the advantage of easily removing the catalyst from reaction media and reusing it.