Objective：This study aims to establish a stable cell line with high expression of anti-plague F1 chimeric antibody，express and purify the chimeric anti-plague F1 antibody and identify its activity. Methods：Total RNA of murine hybridoma cell line 4C6 was extracted and reverse transcribed into cDNA. The variable region genes of light chain and heavy chain of anti-F1 antibody were obtained by PCR and the gene synthesis chimeric antibody heavy chain and light chain gene，and cloning in eukaryotic expression vector pMH3 plasmid of transfection cells. CHO - S stable expression is obtained by G418 screening F1 chimeric antibody cell lines. Supernatant was collected and purified after suspension culture. The specific binding ability with F1 antigen was detected by ELISA. The binding ability of chimeric antibody and mouse parent antibody to antigen was compared by competitive ELISA，the antibody affinity was determined，and the antibody properties were analyzed by mass spectrometry. Results：Stable cell lines secreting chimeric antibody against plague antigen F1 were successfully obtained. The antibody could specifically bind to F1 antigen，and the affinity of the antibody was 2.303×10-9 mol/L. Conclusion：In this study，chimeric antibody against plague F1 was successfully prepared，which laid a foundation for subsequent animal experiments and clinical drug development.