Tile-based self-assembly is a simple yet highly efficient bottom-up nanofabrication strategy, which has been widely applied in many fields such as structural DNA nanotechnology. The self-assembly of DNA tile is determined by not only the sequence design, but also the existing nick in the tile, as it would influence the flexibility and the symmetry of the tile. Herein, we systematically investigated the impact of nick on the tile-based assembly of three-dimensional (3D) DNA polyhedra in various conditions. A series of tile concentrations, four different sticky ends, two annealing approaches, buffers with different metal cations, and different molar percentages of intact tiles, were all tested. It was found that the tile concentration and the valence of metal cations played significant roles in how DNA nick influenced the assembly of DNA polyhedra. Besides, when the molar percentage of intact tiles was higher than the threshold of two-thirds, the impact of nick on the assembly of DNA polyhedra would not be obvious. Our work provides an insightful understanding of the role of nick in the fabrication of high-quality structures from single-tile-based assembly.