DNA is a vector for the genetic information of organisms. It is important to study the interaction between drugs and DNA and to explore its reaction mechanism, the design and synthesis of new drugs. The dissolution behavior of aconitine in water was studied for the first time by microcalorimetry. By using isothermal titration calorimetry, UV-Vis Absorption Spectroscopy and molecular simulation, the interaction between aconitine and armyworm DNA, salmon sperm DNA and calf thymus DNA was discussed. The results showed that the dissolution process of aconitine in aqueous solution was a simple first-order reaction and t1/2=0.691 h. There were two types of combinations during interactions between aconitine and the three different DNAs. The type Ⅰ combination was found to bean enthalpy-driven process. The equilibrium constant of combination (Ka1) was larger with the value of about 105. The number of binding sites (n1) was 0.40~0.60. The reaction took place in the groove areas. With regard to type II combination, the values of Ka2 was 103, and n2 was larger than n1. Lower equilibrium constant of combination demonstrated that the drug ligands just reacted with DNA on the surface instead of entering into the interior of macromolecules. Molecular simulation studies showed that the combination of aconitine and three DNAs occurred in the groove areas. The hydrogen bondwas mainly interactions during interactions between aconitine and the three different DNAs, and the ester groups on the C8 of the aconitine molecule were related to the armyworm DNA, the salmon sperm DNA and the calf thymus DNA respectively. Their bases were specifically identified by T33, T34 and G16, C9, C8. The similarity between the calculated binding energy and the measured ΔG demonstrated that the theoretical calculations were consistent with the experimental results. It can be seen that there is good agreement between the theoretical calculations and experimental results in this work.