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In our previous work, a method of pretreatinglipase was developed to prevent loss of its activity duringcovalent imobilization. In this study, Rhizopus oryzae lipasewas pretreated before imobilization and then imobilizedon a silica gel surface. The effects of the various materials andconditions used in the pretreatment stage on the activity ofpretreated with 0.1% of soybean oil had better activity thanthose pretreated with other materials. The optimal temperature,agitation sped, and pretreating time for lipase pretreatmentwere determined to be 40oC, 200 rpm, and 45 min, respectively.The activity of imobilized soybean oil pretreated lipase was630 U/g matrix, which is 20 times higher than that ofimmobilized non-pretreated lipase. In addition, immobilizedoriginal activity after 10 reuses.