Citrate-coated gold nanoparticles (AuNPs) were prepared by using redox method and its characterization was studied thoroughly. In the preparation process, the varieties of factors were examined by TEM morphology, DLS zetasizer, XPS analysis and UV-Vis spectroscopy for evaluating their qualities and stability. The experimental results obtained under optimal condition, which are 100±5°C of reaction temperature, 4.5-6.4 of the pH value, 2-10 of molar ratio between trisodium citrate and tetrachloroauric acid, respectively. The fluorescent intensities of DCFDA-stain indicate the amount of generated ROS (reactive oxygen species) by AuNPs. The cell cycle for MDA-MB-231 human breast cancer cells exhibits S-phase arrest which can prevent mitosis. The radiosensitizing effect of AuNPs with 20 nm diameter in MDA-MB-231 was investigated by RCGG values (ratio of cell numbers in the G2/M phase to the G0/G1 phase, RCGG) and its radiosensitizing efficiency was ca. 2.29 to 3.75 fold. In this study, it was found that the radiosensitizing efficiency is related to the cytotoxicity by AuNPs. After the irradiation by x-ray to cells, the enhancement value in the presence of 20 nm diameter AuNPs were 17.2% for 3 Gy and 18.9% for 7 Gy respectively. Compared to the irradiated group alone, the enhancements values were 21.8% and 19.7, respectively.