Ternary complex Langmuir-Blodgett (LB) films containing 1,1'-dioctadecyl-4,4'-bipyridinium (SV), deoxyribonucleic acid (DNA), and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphine tetra(p-toluenesulfonate) (TMPyP) were assembled in two ways. One was assembled by depositing the complex monolayer of SV/DNA/TMPyP, which was in situ formed through the adsorption of aqueous subphase containing DNA and TMPyP onto the spreading SV monolayer (type I). Another was obtained by reacting the preformed SV/DNA LB film with TMPyP in aqueous solution (type II). A series of methods, such as surface pressure-area isotherms, atomic force microscopy (AFM), polarized UV-vis spectra, circular dichroism (CD), X-ray diffraction (XRD), and fluorescence lifetime measurements, were used to characterize these ternary LB films. The isotherm measurement indicated that stable complex monolayers could be formed at the air-water interface through the adsorption between SV and DNA or DNA/TMPyP. AFM showed different morphologies of type I and type II LB films. Through a series of characterizations on the LB films, it has been found that TMPyP can intercalate and electrostatically bind to DNA and orient differently in type I and type II LB films. Fluorescence lifetime measurements of the LB films indicated that in the type I LB film electron and/or energy transfer occurred predominantly between the SV and intercalated TMPyP, while in the type II LB film the transfer was predominantly between the SV and electrostatically bound TMPyP. Research showed that the deposition sequence or fabrication method plays an important role in the assembling of ternary LB systems. This method provides a way to fabricate DNA-based molecular assemblies in a controlled and precise manner.