Multilayer films comprising of glucose oxidase (GOx) and phenylboronic acid-modified poly(amidoamine) dendrimer (PBA-PAMAM) were prepared and their pH- and glucose-induced decomposition was studied. Multilayered GOx/PBA-PAMAM films were prepared through boronate ester linkages between PBA-PAMAM and carbohydrate chains of GOx. In contrast, preparation of multilayer film was unsuccessful when unmodified PAMAM was used in lieu of PBA-PAMAM, confirming an essential role of PBA residues in constructing multilayer films. The ten-layered (GOx/PBA-PAMAM)10 film was stable in aqueous solutions of pH 8.5, whereas the multilayer film decomposed in neutral and acidic media as a result of cleavage of the boronate ester bond. The pH threshold for the film decomposition was pH 7.0–8.0. In addition, the (GOx/PBA-PAMAM)10 film decomposed in glucose solutions owing to the oxidative scission of carbon–boron bond of PBA-PAMAM by enzymatically produced hydrogen peroxide. Thus, the potential use of the (GOx/PBA-PAMAM)10 film for the development of pH- and glucose-sensitive devices is suggested.