Plasmid DNA vaccination via microneedles (MNs) to the skin represents potential therapeutics for the clinical treatment of viral diseases and for intradermal genetic immunization. In this study, we investigated DNA vaccination against Severe fever with thrombocytopenia syndrome virus (SFTSV) delivered by nano-patterned microneedles (nMNs) for improving conventional MNs vaccination. Since, DNA vaccination via MNs has major weak points such as poor coating efficiency and immunogenicity etc, additional boosting strategy is necessary. Therefore, we constructed nano-patterned microneedles for improving the affinity of stainless steel of nMNs and plasmid DNA vaccine. The results showed that nMNs have improved DNA vaccine loading capacity, as its surface have improved surface hydrophilicity with increased surface/volume ratio. The cytocompatibility analysis also showed higher cell proliferation with the nMNs. Also, the novel nMNs could be highly beneficial for enhancing intradermal delivery of DNA vaccine across the skin barriers. Finally, the mice group vaccinated with SFTSV DNA vaccine via nMNs generated higher level of humoral and cellular immune responses than those via original MNs.