Sustained release of DNA from the surface of materials represents a promising approach to combine the gene therapy and implantable biomaterials. The nonviral chitosan-DNA complexes were incorporated into the multilayer via layer-by-layer deposition with hyaluronic acid (HA). The UV-vis spectroscopy and atomic force microscopy (AFM) results showed the successful construction of the nonviral complex contained multilayers. The complexes were releasable in physiological condition and a sustained release manner was gained when the multilayer was crosslinked. The cell viability test and the gene transfection assay showed that the natural polyelectrolyte-based nonviral complex incorporated multilayer not only had good cytocompatibility, but also possessed the in vitro gene transfection ability. This kind of surface-mediated nonviral complex incorporated multilayer may have great potential in the localized and controlled delivery of DNA in biomedical implants and tissue engineering application.