Communication: The construction of enzyme multilayer films on colloidal particles for biocatalysis is described. The enzyme multilayers were assembled on submicrometer-sized polystyrene spheres via the alternate adsorption of poly(ethyleneimine) and glucose oxidase using a layer-by-layer approach. Microelectrophoresis and single particle light scattering measurements revealed regular and step-wise assembly of the multilayers on the colloids. The high surface area bio-multilayer coated particles formed were subsequently utilized in enzymatic catalysis.