The electrochemical behavior of o-nitrophenol was studied in detail with a glassy carbon electrode (GCE). The dependence of peak potential on pH indicated that equivalent electrons and protons were involved in the process of o-nitrophenol reduction. The interaction of o-nitrophenol with calf thymus DNA was investigated by adding DNA to the o-nitrophenol solution and by immobilizing DNA on GCE, respectively. The peak current decrement and peak potential shift in presence of DNA indicated that o-nitrophenol could interact with DNA. The result was demonstrated that the in situ DNA damage was detected by differential pulse voltammetry after the o-nitrophenol was electrochemically reduced.