A novel ultrathin enzymatically degradable multilayered film using DNA as building blocks was fabricated by the layer-by-layer (LbL) technique. The UV-vis spectrometry and AFM experiments showed that the buildup of DNA and poly-L-lysine (PLL) was a kind of "exponentially growing films". The dye adsorption experiment suggested that the DNA molecules in the multilayered films were effectively protected by PLL. The films were further cross-linked by glutaraldehyde (GA). The cross-linking density of the films was modulated through the simple controlling of the time of the GA incubation process. An in vitro enzymatic degradation was carried out to investigate the DNA release profiles. The UV-vis spectrometry and fluorescence measurements indicated that the DNA release profiles were accordingly changed with the cross-linking density of the films. The nanoscale, easily processed enzymatically biodegradable PLL/DNA film with the ability to precisely control DNA release profiles may serve as a novel DNA delivery system, which may have great potential for gene therapy applications in implantable materials and biomedical devices.