The hydrolysis kinetics of olive oil dissolved in i-octane with lipase in water was investigated in a biphasic system under various conditions using a Lewis-type transfer cell. The formation rates of fatty acids were found to be affected by the volume of the aqueous solution and the concentrations of lipase and olive oil. The rates followed a reaction scheme in which either the decomposition of a complex on the interface or the desorption of fatty acids formed on the interface into the organic solution was postulated as the rate-determining step. Parameters for the interfacial reaction rate and adsorption equilibrium of lipase were determined so as to satisfy the experimental result.