In the aqueous phase, ethidium bromide (EB) intercalates into the double helix structure of dsDNA (ds=double-stranded) with a notable enhancement in fluorescence and resonance light scattering (RLS). However, when dsDNA was extracted into an ionic liquid (IL), 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF(6)), an abnormal RLS arising from the interactions of IL-DNA-EB was observed, with a substantial decrease of the recorded RLS. The cationic Bmim(+) groups of BmimPF(6) intercalate into the DNA helix structure, in which they interact with the P-O bonds of phosphate groups in DNA strands and result in a reduction of the base-pair interstice along with transformation of DNA conformations that consequently prohibits the intercalation of EB with DNA. Thus, in the IL phase, the interactions between ethidium and DNA were dominated by electrostatic interactions and hydrogen bonding, leading to a congregation of EB entities around the DNA strands that results in an increase of absorption by ethidium, and consequently the inner filter effect leads to a reduction of the RLS. The present observation has been applied to the direct quantification of DNA in an ionic-liquid phase after DNA from human whole blood was extracted into BmimPF(6).