Separate molecular layers of glucoamylase (GA) and glucose oxidase (GOD) were assembled on an ultrafilter by layer-by-layer adsorption using poly(ethyleneimine) (PEI) as a polycation. An aqueous starch solution was placed on the ultrafilter which was covered by GA and GOD layers in various sequential arrangements with intervening polyion complex layers of PEI and poly(styrenesulfonate) (PSS). GA hydrolyzed starch to glucose, which was then converted to gluconic acid and H2O2 by GOD. The films with an upper GA layer and a lower GOD layer showed higher overall activities, consistent with the order of the sequential enzymatic reactions. Reaction products and unreacted starch were readily separated by filtration when the substrate solution was passed through these multi-enzyme films.