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In this study, the extract of coffee powder for green synthesis of zinc oxide nanoparticles has been used because it is compatibility with the environment and it does not produce any toxic substances in the reaction. Then, the interaction of zinc oxide nanoparticles with calf thymus DNA with various spectroscopic methods such as UV-Visible, fluorescence and circular dichroism (CD) techniques was investigated. UV-Visible data showed that zinc oxide nanoparticles induced denaturation in DNA in a dose-dependent manner at both the room and physiologic temperatures. Extrinsic fluorescence emission of ethidium bromide (EB) results also showed that the increase of zinc oxide nanoparticles concentrations, decreases the emission intensity of EB. This may be the result of the intercalation of nanoparticles. Also, CD data showed that the synthesized zinc oxide caused structural changes in DNA. Finally, according to our results, it can be concluded that zinc oxide nanoparticles can bind with DNA and induce some structural changes in DNA structure.