Two types of multifunctional thin films based on DNA polyion complex intercalated with photofunctional chromophore were fabricated on the 2D-surface. One is monolayer of DNA polyion complex transferred to a glass substrate at the air/water interface, and is intercalated with acridine orange (AO). Another is DNA containing thiol moiety, and is intercalated with AO and formed polyion complex with lipid, and is fabricated on a gold substrate at the solid/liquid interface. Molecular orientation of AO was investigated by surface plasmon resonance, polarized UV and FT-IR spectroscopy. AO intercalated DNA polyion complex film (a) was aligned perpendicular to the DNA long axis. In the case of DNA polyion complex film (b), orientated DNA could be fabricated on the gold substrate through S-Au interaction, and the orientation of DNA was more stable by mixing with lipid to provide polyion complex. AO in DNA polyion complex film (b) was aligned parallel to the substrate.