The formation of composite films of double-stranded DNA and cationic lipid molecules (octadecylamine, ODA) and the hybridization of complementary single-stranded DNA molecules in such composite films are demonstrated. The immobilization of DNA is accomplished by simple immersion of a thermally evaporated ODA film in the DNA solution at close to physiological pH. The entrapment of the DNA molecules in the cationic lipid film is dominated by attractive electrostatic interaction between the negatively charged phosphate backbone of the DNA molecules and the protonated amine molecules in the thermally evaporated film and has been quantified using quartz crystal microgravimetry (QCM). Fluorescence studies of DNA-ODA composite films obtained by sequential immersion of the ODA matrix in the complementary single-stranded DNA solutions using ethidium bromide intercalator clearly showed that the hybridization of the DNA single strands had occurred within the composite film. Furthermore, fluorescence studies of the preformed double-stranded DNA-ODA biocomposite film indicated DNA entrapment without distortion to the native double-helical structure. The DNA-ODA biocomposite films have been further characterized with Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) measurements. The DNA-fatty lipid composite films would serve as model systems for understanding DNA-membrane interactions as well as in the study of DNA-drug/protein interactions. This approach also shows promise for the synthesis of patterned DNA films and consequent application in disease detection and genome sequencing.