We developed a multi-enzyme system to enhance the biosynthesis of inositol by covalently immobilizing four purified enzymes on the polyacrylamide hydrogel microparticles. The four purified enzymes form an in vitro enzymatic pathway that converts starch to inositol. Employing a continuous flow column reactor, inositol was produced steadily for 360 hours with the continuous supply of maltodextrin. To examine the effect of the enzyme immobilization on biosynthesis of inositol, the four-enzyme system, either immobilized in hydrogel microparticles or in solution phase, was encapsulated in a continuous flow membrane reactor and continuously catalyzed the conversion of maltodextrin to inositol. The conversion reaction using the multi-enzyme system in the hydrogel lasted for 72 hours longer than the reaction in solution phase. The results showed that immobilization of the multi-enzyme system on the hydrogel polymer backbone substantially improved the function lifetime and the tolerance of enzymes to different pH and high temperature. We expect the immobilized multi-enzyme system combined with the continuous flow reactor provides an economical approach for biosynthesis.