Abstract <p>Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral infectious disease caused by a novel <i>Bandavirus</i> in the family <i>Phenuiviridae</i>. The SFTS virus (SFTSV) is transmitted to various hosts, including humans, through tick bites, leading to high fever, thrombocytopenia, and leukopenia, with a high case fatality rate (up to 30%) due to multiple organ dysfunction. Therefore, early diagnosis is crucial for effective treatment and preventing disease transmission. In this study, we aimed to develop and characterize monoclonal antibodies targeting the SFTSV nucleocapsid protein (NP). We generated recombinant NP to screen antibodies against SFTSV. Using phage display technology, we identified candidate single-chain variable fragment (scFv) sequences capable of detecting SFTSV NP. Five human IgG antibodies and six chimeric mouse antibodies exhibited strong binding ability to the recombinant NP. Furthermore, their specificity and selectivity were evaluated against NPs from different subtypes and other viral species. A sandwich enzyme–linked immunosorbent assay (ELISA) was performed to determine optimal antibody pairings for SFTSV detection. The mP01A05/hP01C09, mP01A05/hP01B10, and mP02E04/hP01A05 antibody pairs demonstrated high efficacy in diagnosing SFTSV infections. These findings provide valuable antibody resources and establish an effective platform for the diagnosis of SFTS.</p> Key points <p><i>• Monoclonal antibodies targeting SFTSV NP were developed using phage display technology.</i></p> <p><i>• Candidate antibodies showed strong binding ability and high specificity to SFTSV NP.</i></p> <p><i>• Optimized antibody pairs enabled effective SFTSV detection via sandwich ELISA.</i></p>