Abstract: The interaction between tritiated water and λ double-stranded deoxyribonucleic acid(λ-dsDNA) was investigated using Raman spectroscopy and density functional theory(DFT). Raman spectroscopy was applied to analyse structural changes in λ-dsDNA after HTO of different radioactivity(1×10⁹ Bq/L and 1×10¹¹ Bq/L) and γ-irradiation(50 mGy to 8 Gy). At a low dose(100-500 mGy) of 1×10⁹ Bq/L HTO is mainly indirect effect of ionizing radiation during irradiation, destroying hydrogen bonds between bases caused base mismatches and modification in base structure, resulting in better unpairing of base pairs. However, 1×10¹¹ Bq/L HTO on λ-dsDNA in low dose(50-100 mGy) for a short period of time is similar to γ-ray, mainly caused by direct ionization or protonation of the ray, which leads to the destruction of the base structure. In addition, high radiation dose(2-8 Gy)  of HTO can cause the conformational transfer of the furanose ring or the cleavage of the covalent bond through tritium-hydrogen(T-H) exchange reaction. It is observed by spectral markers that the three modes of action of HTO may eventually lead to changes in λ-dsDNA backbone conformation and λ-dsDNA denaturation.