In the recent years, the microscopic magnetic resonance technology using nitrogen-vacancy (NV) centers in diamond as quantum sensors has been developed rapidly. The detection of magnetic resonance spectrum of a single biomolecule has been achieved, and it now steps to the study of single molecule’s structure and function. Therefore, it is necessary to solve an important technical problem: how to disperse and fix biomolecules on diamond surface orderly. DNA self-assembly provides a possible way to solve this problem. In this paper, 60 nm square double-layer DNA origami was prepared as a kind of single-molecule carrier and then combined to the diamond surface. Firstly, we used the double layer structure to enhance the DNA origami’s strength. Secondly, by adding hairpin structure to the edge of DNA origami, the agglomeration between DNA origami was reduced. Finally, the DNA origami was successfully assembled to the diamond surface. The atomic force microscope images showed that the structure was complete and the dispersion was uniform. This work built a foundation for the subsequent application of single-molecule magnetic resonance technology in the field of biophysics.