The characterization of a biosensor based on poly(allylamine) containing an osmium complex (OsPAA) in a matrix of industrial relevance, like milk, is presented. The biosensor is constructed by crosslinking an oxidase and the redox polymer (OsPAA) with poly(ethylene diglycidyl) ether (PEG). Parameters such as the optimum crosslinker concentration, working pH and temperature, and the effect of ionic strength were examined. When the electrode was immersed in milk spiked with substrate, an important decrease in the amperometric signal was observed compared with the one observed in a buffer solution at pH 7.0. The effects of different components of the milk are presented. The decay in the catalytic current is mainly due to the caseins, which insulate the osmium complex from the redox center of the oxidase.