A stable, biocompatible single strand DNA (ssDNA)/bovine serum albumin (BSA) multilayered film for control release of DNA was fabricated on PEI-coated quartz slides, gold-evaporated plates and silicon wafers, respectively through a formaldehyde-induced, covalently linked layer-by-layer (LBL) assembly technique. The constructed film structure was well characterized by using UV-vis spectrometry, surface plasmon resonance (SPR) and atomic force microscopy (AFM). The results showed that the DNA incorporated LBL film was fabricated successfully and the amount of ssDNA and BSA in the film could be tailored simply by controlling the number of the bilayers. The control release of DNA from the film was also monitored in this study. UV-vis spectrometry, SPR and AFM measurements indicated that the release of ssDNA and amino acid was adjustable by changing the proteinase K incubation time. This biocompatible covalently assembled film demonstrates an innovative approach to engineer a DNA/protein based nanostructure for controlled DNA release, which could provide stability, controllability and flexibility superior to that of LBL film assembled by electrostatic attraction. Since the film in this work can be assembled on different substrates, it is very feasible to fabricate nanoparticle-based gene therapy systems with this new approach and to have great potential in biomedical applications. (c) 2007 Elsevier Inc. All rights reserved.