In this work, a novel biosensor was fabricated for detection of DNA damage induced by 4-nonylphenol (NP) and also determination of NP. To achieve this goal, a glassy carbon electrode (GCE) was modified with chitosan (Chit), gold nanoparticles (Au NPs) and DNA-multiwalled carbon nanotubes (DNA-MWCNTs). Then, the DNA-MWCNTs/Au NPs/Chit/GCE was incubated with methylene blue (MB) to obtain MB-DNA-MWCNTs/Au NPs/Chit/GCE in which MB was used as the redox indicator. The modifications applied to the GCE were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopic (EDS) and theoretical evidence. MB is a derivative of anthraquinone which can intercalate into double helix structure of DNA. By treating MB-DNA-MWCNTs/Au NPs/Chit/GCE with NP, a higher R ct was observed because the insertion of the NP may result in a more negative charge environment on the DNA surface which hinders accessibility of [Fe(CN) 6 ] 3-/4- anion to the electrode surface. Change in the EIS response of the biosensor in the presence of NP was used to develop a novel system for monitoring the DNA damage induced by NP. The EIS technique was also used to develop a sensitive electroanalytical method for determination of NP., (© 2019 The Authors. Published by Elsevier Ltd.)