The interaction of anticancer drug irinotecan (CPT-11), which is the inhibitor of the Topoisomerase I enzyme, with fish sperm double stranded deoxyribonucleic acid (dsDNA) and synthetic short oligonucleotides were studied electrochemically based on the oxidation signals of guanine and CPT-11 by using differential pulse voltammetry (DPV) and cyclic voltammetry (CV) at pencil graphite electrode (PGE). In this work, three types of methods, such as adsorption, covalent attachment and electrostatic binding were used for the immobilization of DNA onto the PGE surface. It is found that an effective modification method for DNA on the electrode surface is very important because it effects the drug and DNA interaction. As a result of the interaction, the electrochemical signal of guanine and CPT-11 greatly decreased. Experimental parameters, such as the effect of buffer solution on the interaction between CPT-11 and DNA, the concentration of CPT-11/DNA, the immobilization time of DNA and the accumulation time of CPT-11 were studied in DPV; in addition, the interaction of CPT-11 with oligonucleotides was evaluated for using as a hybridization indicator in CV and DPV. The detection limit and the reproducibility were also determined.