Dinitramine is a herbicide that has been used to control annual grasses and broadleaf weeds in cotton and soybeans in Iran. In this study, the electrochemical behavior of dinitramine was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. The interaction of dinitramine with ct-DNA was evaluated by CV, competitive fluorescence, UV-Vis spectroscopy, FT-IR spectroscopy, and viscosity titration. In addition, the thermodynamic parameters of DIN-DNA complex were calculated by spectrophotometric titration. The values of ΔH bin. , ΔS bin., and ΔG bin. (T = 290.65 K) of the DIN-DNA complex were +39.25 kJ mol −1 , +215.71 J mol −1, and −23.45 KJ mol −1 , respectively. These data revealed that the endothermic binding has its origin in the hydrophobic interactions. Also the high positive ΔS bin was explained according to the DIN structure that optimized by mechanical quantum calculations. However, all data showed that the major groove binding between DIN and ct-DNA is more predominant than other binding modes.