Gold nanoparticles have been shown to enhance radiation doses delivered to biological targets due to the high absorption coefficient of gold atoms, stemming from their high atomic number (Z) and physical density. These properties significantly increase the likelihood of photoelectric effects and Compton scattering interactions. Gold nanoparticles are a novel radiosensitizing agent that can potentially be used to increase the effectiveness of current radiation therapy techniques and improve the diagnosis and treatment of cancer. However, the optimum radiosensitization effect of gold nanoparticles is strongly dependent on photon energy, which theoretically is predicted to occur in the kilovoltage range of energy. In this research, synchrotron-generated monoenergetic X-rays in the 30–100 keV range were used to investigate the energy dependence of radiosensitization by gold nanoparticles and also to determine the photon energy that produces optimum effects. This investigation was conducted using cells in culture to measure dose enhancement. Bovine aortic endothelial cells with and without gold nanoparticles were irradiated with X-rays at energies of 30, 40, 50, 60, 70, 81, and 100 keV. Trypan blue exclusion assays were performed after irradiation to determine cell viability. Cell radiosensitivity enhancement was indicated by the dose enhancement factor which was found to be maximum at 40 keV with a value of 3.47. The dose enhancement factor obtained at other energy levels followed the same direction as the theoretical calculations based on the ratio of the mass energy absorption coefficients of gold and water. This experimental evidence shows that the radiosensitization effect of gold nanoparticles varies with photon energy as predicted from theoretical calculations. However, prediction based on theoretical assumptions is sometimes difficult due to the complexity of biological systems, so further study at the cellular level is required to fully characterize the effects of gold nanoparticles with ionizing radiation.