Four configurations of an enzyme electrode to assess lactate concentration in dairy products are studied. In these bienzymatic multilayer systems, the inner layer was constructed by crosslinking peroxidase with poly(allylamine) containing an osmium complex (OsPAA), and an outer layer containing lactate oxidase (LOx) immobilized in polymeric matrices. When the hydrogen peroxide formed by the LOx layer reaches the inner layer, the electronic flow between the wired peroxidase and the electrode surface produces a cathodic current, proportional to lactate concentration. Parameters such as the effect of ionic strength, stability when the electrode is used as detector in a FIA system and reproducibility were examined. This arrangement allows working in undiluted milk samples.