Objective:To prepare monoclonal antibodies specific to human papillomavirus type 6 L1 (HPV6 L1) protein, investigate the binding characteristics, cross-reactivity, and neutralization efficacy of these antibodies.Methods:Antibody genes were extracted from the spleen cells of mice immunized with HPV6 L1 protein and used to construct an ScFv phage antibody library. The library was enriched and screened using HPV6 L1 protein. Following expression of the antibody genes into murine IgG, the binding properties of the antibodies to the L1 protein and their neutralization effects on pseudoviruses were verified.Results:An ScFv antibody library targeting HPV6 L1 was successfully constructed with a capacity of 6.54×10 7. After screening with HPV6 L1, two antibody strains were obtained, named Q9 and Q11. ELISA testing revealed that Q9-IgG has a high antibody titer (about 10 6) against HPV6 L1, while cross-reactivity titers to HPV18 and 45 L1 were 10 2. Q11-IgG has an antibody titer about 4×10 4 against HPV6 L1, but not combined with the L1 proteins of other HPV types. Pseudovirus neutralization assays indicated that Q9-IgG lacked neutralizing activity, while Q11-IgG showed a neutralizing antibody titer of 10 4.5against HPV6 pseudovirus. Conclusions:Both Q9 and Q11 are monoclonal antibodies specific to HPV6 L1. However, they displayed notable differences in cross-reactivity, antigen recognition sites, and neutralization efficacy. These antibodies provide essential tools for the foundational research of HPV6, immunological diagnosis, and the development of therapeutic formulations.