A detailed understanding of the interactions between biomolecules and nanomaterial surfaces is critical for the development of biomedical applications of these nanomaterials. Here, we characterized the binding patterns and dynamics of a double stranded DNA (dsDNA) segment on the recently synthesized nitrogenized graphene (C2N) with both theoretical (including classical and quantum calculations) and experimental approaches. Our results show that the dsDNA repeatedly exhibits a strong preference in its binding mode on the C2N substrate, displaying an upright orientation that is independent of its initial configurations. Interestingly, once bound to the C2N monolayer, the transverse mobility of the dsDNA is highly restricted. Further energetic and structural analyses reveal that the strength and position of the binding is guided by the favorable stacking between the dsDNA terminal base pairs and the benzene rings on the C2N surface, accompanied by a simultaneous strong nanoscale dewetting that provides additional driving forces. The periodic atomic charge distributions on C2N (from its unique porous structure) also cause the formation of local highly dense first solvation shell water clusters, which act as further steric hindrance for the dsDNA migration. Furthermore, free energy profiling calculated by the umbrella sampling technique quantitatively supports these observations. When compared to graphene, C2N is found to show a milder attraction to dsDNA, which is confirmed by experiments. This orientational binding of DNA on the C2N substrate might shed light on the design of template-guided nanostructures where their functions can be tuned by specialized biomolecular coating.