<p>Multiple outbreaks of Ebola virus in West Africa have posed significant threats to global public health owing to its high pathogenicity and fatality rates. Current treatments for Ebola Virus Disease are limited, underscoring the imperative for novel antiviral therapies. VP30, a critical RNA synthesis factor, interacts with nucleoprotein (NP) to facilitate Ebola viral genome transcription and replication. Notably, the host ubiquitin-ligase retinoblastoma-binding protein 6 (RBBP6) binds to VP30 at the same interface as NP, thereby inhibiting VP30-NP interactions and indicating that targeting this interface could advance antiviral drug development. In this study, we engineered six peptide mutants through amino acid substitutions at key VP30 binding sites. These mutants were fused to DNA-binding protein from starved cells 4 (DPS4) to assemble nanoparticles, enabling surface display of the peptides. Antiviral effects were evaluated using minigenome and transcription and replication-competent virus-like particles (trVLPs) systems. Among the variants, RPL1 and NPL3 peptides exhibited relatively strong apparent affinities with the VP30 and potent antiviral activity by disrupting Ebola viral genome transcription and replication. To elucidate the binding details between the peptides and VP30, we determined crystal structures of complexes between RPL1 or NPL3 peptides and VP30 via X-ray crystallography. Concurrently, molecular dynamics (MD) simulations revealed the dynamic binding processes of these peptides to VP30. Structural analyses confirmed that the peptides bind to the VP30/NP interface and compete with NP. Our findings demonstrate that DPS4-fusion peptides effectively deliver peptides into cells as nanoparticles and inhibit VP30-NP interactions, presenting a novel antiviral strategy for Ebola virus.</p> Graphical abstract <p></p>