A new electrochemical biosensor was developed to demonstrate the effect of Acridine Orange (AO) on DNA damage. Then, the biosensor was used to check the inhibitors effect of three flavonols antioxidants (myricetin, fisetin and kaempferol) on DNA damage. Acridine Orange (AO) was used as a damaging agent because it shows a high affinity to nucleic acid and stretch of the double helical structure of DNA. Decreasing on the oxidation signals of adenine and guanine (in the DNA) in the presence of AO were used as probes to study the antioxidants power, using DNA-modified screen printed graphene electrode (DNA/ SPGE). The results of our study showed that the DNA-biosensor could be suitable biosensor to investigate the inhibitors ability of the flavonols antioxidants on the DNA damage. The linear dependency was detected in the two regions in the ranges of 1.0e15.0 and 15.0e500.0 pmol L1. The detection limit was found 0.5 pmol L 1 and 0.6 pmol L1 for guanine and adenine, respectively. To confirm the electrochemical results, UveVis and fluorescence spectroscopic methods were used too. Finally molecular dynamic (MD) simulation was performed on the structure of DNA in a water box to study any interactionbetween the antioxidant, AO and DNA.