The immobilization and biocatalysis of commercially purified soybean lipoxygenase (LOX) type I-B (EC 1.13.11.12) were investigated in organic solvent media. The results showed that the highest immobilization efficiencies of LOX, 30.6 and 29.3%, were obtained with DEAE-cellulose and modified Eupergit (R) C250L supports, respectively. The biocatalysis of free and immobilized (Eupergit (R) C25OL/EDA) LOXs was investigated in different mixtures of hexane and a selected cosolvent (95:5 [v/v]). The results showed a 1.5 and a 1.6 increase in the activity of free and immobilized LOXs, respectively, using a mixture of hexane and 1,4-dioxane compared with that in hexane alone; however, cosolvents, including 2-octanone, 2-heptanone, 2-butanone, and cyclohexanone, displayed an inhibitory effect on LOX activity. In the mixture of 1,4-dioxane and hexane, LOX activity was dependent on the cosolvent concentration, which was increased with 1,4-dioxane up to 5% (v/v). The threshold 1,4-dioxane concentration (C-50) and the incubation period (T-50) at which 50% of the maximal enzyme activity was obtained for the free and immobilized LOXs were 6.7 and 8.9% (v/v) and 9.1 and 17.0 min, respectively.