Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) ; Control of supramolecular architectures inherent in layer-by-layer (LbL) films allows far synergistic effects be, tween the film components, as in reactions under controlled conditions. In this paper we show that a bienzyme system can be produced in LbL films, in which the product of a reaction catalyzed by invertase (INV) is used in a catalysis process involving glucose oxidase (GOx). As a proof of principle, simultaneous detection of glucose and sucrose is demonstrated for an optimized film architecture comprising poly allyllamine hydrochloride (PAH) alternated with INV + GOx layers (PAH/(GOx + INV))(10). Though the sensitivity for sucrose is low because the product from the reaction with invertase has low electroactivity, the results confirm that cascade reactions can be performed in a controlled manner in LbL films with possible applications in sensors and bioelectrochemical devices.