Supramolecular crystals arise from noncovalent interactions between macromonomers and allow for the engineering of dynamic functional materials. For two-dimensional (2D) crystals, the substrate surface can induce the formation of new polymorphs not available in solution, adding a layer of complexity to the supramolecular self-assembly process. Despite extensive studies on the 2D self-assembly of supramolecular crystals, unknowns remain regarding substrate-monomer interactions and the effects on network self-assembly and defect repair. Here, we used a DNA-mica model system to modulate and understand the impact of substrate-monomer interactions on the crystalline order. We controlled the surface interactions by tuning the Mg2+ concentration, varying the divalent cation type, and adjusting the relative concentration of divalent and monovalent cations. The competition between monovalent and divalent cations yielded nearly defect-free crystals with minimal polygon defects. These findings highlight the critical role of surface interactions in achieving high crystalline order, which is essential for optimizing the efficiency and performance of supramolecular functional nanomaterials.