Geotrichum sp. lipase with enhanced activity and operational stability was prepared for use in nonaqueous media. A combined strategy comprising bioimprinting with dual imprint molecules and a cosolvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum sp. lipase. The modified lipase exhibited 18.4-fold enhanced esterification activity towards methyl oleate synthesis, and retained 90% activity following repeated use in 10 cycles. The combined strategy exhibited a significant synergistic effect and was suitable for lipase modification, dramatically enhancing the enzyme activity and operational stability. This approach is applicable to the preparation of other enzyme biocatalysts, since the methods are effective for upgrading crude enzyme to a refined product with high activity and stability for use in non-aqueous media. (C) 2009 Elsevier Ltd. All rights reserved.