The detection of DNA damage is one of the most important topics in the DNA research fields. In this paper, an electrochemical method for the detection of DNA damage by combining the layer-by-layer assembly film with adriamycin (ADM) as an electrochemical probe was developed. Firstly, the layer-by-layer {dsDNA/PEI}(n) film was prepared by the alternate adsorption of polycationic polyethyleneimine (PEI) and negatively charged natural DNA onto glassy carbon electrode (GCE) surface. Its electrochemical behaviors were characterized by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV), and the optimal value of n was determined to be 2. Secondly, the {dsDNA/PEI}(2) film was immersed into the solution of ADM and the {dsDNA/PEI}(2)-ADM was obtained. While transferred into the blank solution, the {dsDNA/PEI}(2)-ADM would gradually release ADM, exhibiting the good reversibility of ADM incorporation. Finally, the DNA damage induced by styrene oxide (SO) was investigated and the promising results showed that the present method can be a useful tool for the detection of DNA damage.