Epstein-Barr Virus (EBV) is considered the most important human pathogen due to its role in infections and cellular malignancies. It has been reported that this Oncolytic virus infects 90% world’s population. EBNA1 is required for DNA binding and survival of the virus and is considered an essential drug target. The biochemical and structural properties of this protein are known, but it is still unclear which residues impart a critical role in the recognition of dsDNA. Intending to disclose only the essential residues in recognition of dsDNA, this study used a computational pipeline to generate an alanine mutant of each interacting residue and determine the impact on the binding. Our analysis revealed that R469A, K514A, Y518A, R521A and R522A are the key hotspots for the recognition of dsDNA by the EBNA1. The dynamics properties, i.e. stability, flexibility, structural compactness, hydrogen bonding frequency, binding affinity, are altered by disrupting the protein-DNA contacts, thereby decreases the binding affinity. In particular, the two arginine substitution, R521A and R522A, significantly affected the total binding energy. Thus, we hypothesize that these residues impart a critical role in the dsDNA recognition and pathogenesis. This study would help to design structure-based drugs against the EBV infections.