The fluorescence spectra in combination with UV–Vis absorption were used in the present work. The binding of quercetin and rutin flavonoids with DNA was investigated in Tris-HCl buffer (pH = 7) by using acridine orange dye. A strong fluorescence quenching reaction of DNA-acridine orange dye to these flavonoids was observed where the dye has an emission band at 528 nm after excitation at 490 nm. The binding constant was determined to be 2.99x10⁷, 4.14x10² mol^-1L in case of quercetin and rutin, respectively. The type of interaction was determined to be electrostatic for quercetin and hydrophobic for rutin. The binding constant of arginine, asparagine and phenylalanine amino acids with DNA was determined to be 1.8x10², 8.71x10³ and 3.61x10² mol^-1L respectively. The interaction of quercetin and rutin with DNA–amino acid complexes were studied where the results indicate that binding of these amino acids with DNA has significant effect on increasing the binding constant of the studied flavonoids with DNA, asparagine and phenylalanine has an effect on converting the type of interaction of quercetin with DNA from electrostatic to van der Waals force or hydrogen bond, and arginine convert the interaction type of rutin with DNA from hydrophobic to van der Waals force or hydrogen bond.