Layer-by-layer assembly of DNA film was fabricated on quartz plates through alternative adsorption with poly(allylamine) in order to investigate the interaction of DNA film with dyes. The film was water-insoluble and can interact with many water-soluble dyes. It has been found that ethidium bromide (EB) and 5,10,15,20-tetrakis(4-N-methylpyridyl)porphine tetra(p-toluenesulfonate) (TMPyP) can intercalate into the DNA film, which has been verified from the UV and fluorescence :spectra and circular dichroism (CD) measurements. Acridine orange (AO) can also interact with the DNA film, but it seems that AO attaches to the DNA as a dimer. It has been further confirmed that TMPyP can predominantly intercalate into the assembled DNA film, while all the dyes coexisted in the aqueous solution. In the case of TMPyP solution only, TMPyP intercalated predominantly into the G-C site, while in the mixture it went mainly into the A-T site of the DNA film.