It has been proposed that the radiation damage to biological particles and soft condensed matter can be overcome by ultrafast and ultraintense X-ray free-electron lasers (FELs) with short pulse durations. The successful demonstration of the “diffraction-before-destruction” concept has made single-shot diffraction imaging a promising tool to achieve high resolutions under the native states of samples. However, the resolution is still limited because of the low signal-to-noise ratio, especially for biological specimens such as cells, viruses, and macromolecular particles. Here, we present a demonstration single-shot diffraction imaging experiment of DNA-based structures at SPring-8 Angstrom Compact Free Electron Laser (SACLA), Japan. Through quantitative analysis of the reconstructed images, the scattering abilities of gold and DNA were demonstrated. Suggestions for extracting valid DNA signals from noisy diffraction patterns were also explained and outlined. To sketch out the necessary experimental conditions for the 3D imaging of DNA origami or DNA macromolecular particles, we carried out numerical simulations with practical detector noise and experimental geometry using the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory, USA. The simulated results demonstrate that it is possible to capture images of DNA-based structures at high resolutions with the technique development of current and next-generation X-ray FEL facilities.