Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree. (C) 2011 Elsevier Ltd. All rights reserved.