Glucose-sensitive films were prepared through the layer-by-layer (LbL) deposition of hemin-modified poly(ethyleneimine) (H-PEI) solution and DNA solution (containing glucose oxidase (GOx)). H-PEI/DNA + GOx multilayer films were constructed using electrostatic interactions. The (H-PEI/DNA + GOx)(5) film was then partially decomposed by hydrogen peroxide (H2O2). The mechanism for the decomposition of the LbL film was considered to involve more reactive oxygen species (ROS) that were formed by the reaction of hemin and H2O2, which then caused nonspecific DNA cleavage. In addition, GOx present in the LbL films reacts with glucose to generate hydrogen peroxide. Therefore, decomposition of the (H-PEI/DNA + GOx)(5) film was observed when the thin film was immersed in a glucose solution. (H-PEI/DNA + GOx)(5) films exposed to a glucose solution for periods of 24, 48 72, and 96 h indicated that the decomposition of the film increased with the time to 9.97%, 16.3%, 23.1%, and 30.5%, respectively. The rate of LbL film decomposition increased with the glucose concentration. At pH and ionic strengths close to physiological conditions, it was possible to slowly decompose the LbL film at low glucose concentrations of 1-10 mM.