Abstract: Following the discharge of nuclear wastewater in Fukushima, the radiation biological effects of tritium have become a focus of public health concern. Tritium readily enters the human body and participates in metabolic activities. Its decay emits β-rays, which pose significant harms to human health. Also, targeted radiotherapy utilizes the ability of ingested Auger electron emitters to enter cancer cells and the release Auger electrons to cause severe damage to DNA, thereby effectively killing cancer cells. To investigate the radiation biological effects caused by β-rays, the nanodosimetry program NASIC developed by Tsinghua University was utilized to calculate the cellular S values across various cell sizes and the yield of double strand breaks (DSB) induced by ¹²⁵I and tritium, delving into their potential radiobiological effects. In this study, the updated ¹²⁵I Auger electron emission spectrum from Howell et al. and the tritium decay electron energy spectrum provided by the U. S. Nuclear Regulatory Commission were adopted to conduct a comprehensive analysis of cellular radiobiological effects. Utilizing the single-cell model endorsed by Medical Internal Radiation Dose (MIRD), the cell nucleus was designated as the primary target and the combined cytoplasm and cell nucleus were the source regions, respectively. The celluar S values, S(N←Cy) and S(N←N), representing the average absorbed dose to the nucleus from radionuclides distributed in the cytoplasm and nucleus respectively, were systematically quantified. Furthermore, a sophisticated random sampling technique was incorporated to delineate DNA interactions within the nucleus. It has been meticulously assessed that the induction rates of DSB caused by isotopically uniform distributions of ¹²⁵I and tritium, both diffusely located throughout the nucleus and directly bonded to the DNA helix. Discrepancies in calculated cellular S values and published data, attributable to varying computational methods and low-energy electron cross-sections, are presented in this study. The S(N←N) derived from the uniform distribution of ¹²⁵I and tritium in the cell nucleus, closely align with the published data, while those from radionuclide distribution in the cytoplasm S(N←Cy) deviate significantly, with the deviation from 4.23% to 26.35%. These discrepancies underscore the sensitivity of radiobiological calculations to the employed computational approaches and cross-sections data, particularly for cytoplasmic distributions. Additionally, the results show that direct binding of ¹²⁵I and tritium to DNA, compared to their uniform distribution in the nucleus, substantially increases the DSB yield: Direct binding of ¹²⁵I results in a DSB yield twice that of the uniform distribution, while tritium binding increases the DSB yield from 1.31×10⁻¹¹ Gy⁻¹·Da⁻¹ to 1.46×10⁻¹¹ Gy⁻¹·Da⁻¹. This study deeply explores the damaging mechanism of radionuclides at the biological micro- and nano scale, providing an important theoretical basis for precision medicine and environmental radioactive contamination risk assessment.