Fraxinellone is an important botanical lactone compound andhas been demonstrated to have insecticidal activity. To provide theoretical support tothe assessment on the safetyof utilizingfraxinellone as a natural insecticidal agent, the interactions between fraxinellone and armyworm DNA, salmon sperm DNA and calf thymus DNA were investigated using UV-Vis absorption spectroscopy, isothermal titration calorimetry, and molecular docking. Results showed that there were two types of combinations between fraxinellone and three kinds of DNA. Type I combination had an equilibrium constant of combination (Ka1) of about 105 and binding sites (n1) of 0.40-0.70, while type II combination had an equilibrium constant of combination (Ka2) of 103 and binding sites (n2) of 1.35-3.15. Results of molecular docking showed that there werenon-classical embedding type interactions between fraxinellone and three kinds of DNA, with the reaction taking place in small groove areas of the DNA structure, resulting in relatively weak interactive forces.