The Epstein–Barr virus (EBV) is one of the cancer-causing gamma-type viruses. Although more than 90% of people are infected by this virus at some point, it remains in the body in a latent state, typically causing only minor symptoms. Our current understanding is that a known transcription factor (TF), the ZTA protein, binds with dsDNA (double-stranded DNA) and plays a crucial role in mediating the viral latent-to-lytic cycle through binding of specific ZTA-responsive elements (ZREs). However, there is no clear understanding of the effect of DNA sequences on the structural stability and quantitative estimation of the binding affinity between ZTA TF and DNA, along with their mechanistic details. In this study, we employed classical all-atom molecular dynamics and enhanced sampling simulations to study the ZTA-dsDNA structural properties, thermodynamics, and mechanistic details for the ZTA protein and for two different dsDNA systems: the core motif and the core motif with flanking end sequences. We conducted residue-level and nucleic acid-level analyses to assess the important protein residues and DNA bases forming interactions between the ZTA and dsDNA systems. We also explored the effect of adding flanking end sequences to the core motif on DNA groove lengths and interstrand hydrogen bonds. Our results indicate that the flanking sequences surrounding the core motif significantly influence the structural stability and binding affinity of the ZTA–dsDNA complex. Among ZRE 1, ZRE 2, and ZRE 3, particularly when paired with their naturally occurring flanking ends, ZRE 3 exhibits higher stability and binding affinity. These findings provide insights into the molecular mechanisms underlying EBV pathogenesis and may indicate potential targets for therapeutic intervention. A detailed explanation of the binding mechanisms will allow for the design of better-targeted therapies against EBV-associated cancers. This study will serve as a holistic benchmark for future studies of these viral protein interactions.