In this study, Candida rugosa lipase was pretreated with soybean oil in order to allow fatty acids to bond to the active site before immobilization. The pretreated C. rugosa lipase was immobilized on β-cyclodextrin-based polymer by crosslinking with hexamethylene diisocyanate (HMDI). This pretreated lipase exhibited steric hindrance around the active site such that during immobilization, covalent bonds were formed between the carrier and the lipase region far from the active site. The pretreated lipase was immobilized on a β-cyclodextrin-based polymer by crosslinking with hexamethylene diisocyanate with the activity of 42 U/g-matrix, which is 5.7 times higher than that of the immobilized non-pretreated lipase. In addition, the immobilized lipase activity was maintained at levels exceeding 60% of its original activity after 10 reuses. © 2008 Elsevier B.V. All rights reserved.