An electrochemical DNA nano-biosensor is prepared by immobilization of double stranded DNA (dsDNA) onto a mixed self-assembled monolayer (SAM) composed of azide- and hydroxyl-terminated thiols. The SAMs- and dsDNA-modified gold electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of spermidine with DNA was studied by differential pulse voltammetry at the DNA-modified electrode. The decrease in the guanine oxidation peak current was used to study the interaction. The binding constant (K), obtained by differential pulse voltammetry, was 1.85×10 5 M −1 . A linear dependence of the guanine peak currents was observed in the range of 1.6–70.4μM spermidine, with a detection limit of 0.72μM and r=0.994 by using differential pulse voltammetry.