Objective:To obtain an anti-enterovirus 71 (EV71)-VP1 monoclonal antibody, and preliminarily analyze its biological characteristics. Methods:Immunized the BALB/c mice with purified recombinant EV71-VP1 protein. The spleen cells were fused with mouse myeloma cells(Sp2/0). Subsequently, limited dilution method was used to screen positive hybridoma cell lines. After several times of subclone, one hybridoma cell line (6F2B9)was obtained. The supematant of 6F2B9 was collected and purified by affinity chromatography with Protein G sepharose. The concentrations of antibodies were tested by BCA method and its subtype was identified by Ig subtype qualitation kit. The titer and specificity of the McAb were detected by indirect ELISA. We used indirect immunefluorescence assay (IFA) to observe specifical combination with EV71 virus. Results:We succeeded in selecting a hybridoma cell line secreting anti-EV71-VP1 monoclonal antibody. The McAb obtained was named DSE-136 and belonged to IgG2b, κ light chain, and its titer was 1∶3.2×105 and could specifically bind to the surface of EV71 which was detected by IFA. Conclusion:We successfully obtained a mouse anti-EV71-VP1 McAb with high dilution and specificity, which has laid foundation on antigen diagnosis and vaccine research and development for EV71.