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The controllable and sustained release of DNA from the surfaces of biomaterials or biomedical devices represents a new method for localized gene delivery. We report the synthesis of a novel polycation containing disulfide bonds in its backbone and the fabrication of polycation/plasmid DNA multilayered thin films by layer-by-layer assembly. The films are very stable during preparation and in storage, however, they gradually degrade and release the incorporated DNA when incubated in PBS buffer containing dithiothreitol (DTT), which results from the degradation of a disulfide-contained polymer under reductive conditions. The film degradation rate and DNA release rate can be tuned by the concentration of reducing agent. This approach will be useful in gene therapy and tissue engineering by controlled administration of therapeutic DNA deposited on the surface of implantable biomedical devices or tissue engineering scaffolds.