In this paper, DNA-based honeycomb films were successfully constructed from a simple solution casting process at high relative humidity, through the encapsulation of DNA with a cationic surfactant ditetradecyldimethylammonium (DTDA). Cationic surfactants electrostatically attach to phosphate anions of DNA in a molar ratio of 1:1 in the DNA-DTDA complex. Investigation of the effects of substrate, concentration, and solvent on the morphology of the microporous films demonstrated the wide generality and high reproducibility of the formation of DNA-DTDA self-organized microporous films. The morphology of the microporous structures can be adjusted by slightly changing the concentration of the complex solution. DNA exists in the double helical B-form in the microporous film confirmed by circular dichroism spectrum, Dye molecule rhodamine B was loaded into the DNA-DTDA complex, presenting a fluorescent microporous film. DNA-DTDA complex as a host material provides a new method for tailoring fluorescent microporous Films. The DNA-based ordered honeycomb films should be attractive for use in optical and optoelectronic devices, separation, ion transportation, and biosensors.