Provided is a method for regenerating an immobilized enzyme for lipolysis which has been used for lipolysis, thereby exhibiting a reduced activity, which includes washing the immobilized enzyme with a solvent; controlling an equilibrium concentration of fatty acids in the solvent; removing the washed immobilized enzyme therefrom, and contacting the resulting immobilized enzyme with a fresh enzyme, wherein the fresh enzyme adsorbs onto the immobilized enzyme. According to the preferred methods of the present invention, it is possible to make effective use of the residual activity of an immobilized enzyme spent for lipolysis and regenerate the spent immobilized enzyme to have a performance similar to that before the lipolysis at a low cost using less amount of liquid waste.