Abstract: 【Objective】This study prepared and indentified monoclonal antibodies(mAbs) against penton protein of fowl adenovirus serotype 4(FAdV-4), in order to further study the function of penton protein in FAdV-4 infection pathogenesis and provide antibody materials for the research and development of antigen detection reagent. 【Method】Female 6-8 weeks old BALB/c mice were immunized with the penton protein of FAdV-4 in prokaryotic expression. After three times of booster immunization, cell fusion was performed with spleen cells and SP2/0 myeloma cells of mice. Positive hybridoma cell lines were screened by penton-enzyme linked immunosorbent assay(ELISA) and FAdV4 virus-ELISA respectively. Subclass identification, specific detection, broad-spectrum detection, indirect immunofluorescence and stability tests were performed for the screened hybridoma cell lines against monoclonal antibodies. 【Result】Resultsof subclass identification showed that seven hybridoma cell lines that could stably secrete penton protein against FAdV-4(1D11, 2B3, 2C4, 2C9, 6B3, 8F12 and 8G11) have been screened. Light chains of seven hybridoma cell lines belonged to Kappa chain, three IgG1 heavy chains and four IgG2b heavy chains.The results showed that seven monoclonal antibodies only reacted with FAdV-4, and did not cross-react with other 11 serotypes FAdV, NDV, ARV, EDSV, IBV and other common avian pathogens. The results of broad spectrum identification showed that all seven monoclonal antibodies could bind to 19 FAdV-4 isolates with good broad spectrum. Indirect immunofluorescence assay showed that seven monoclonal antibodies were bound to FAdV-4 infected chick embryonic hepatocytes(CEL) and showed bright green fluorescence.The stability test results showed that three strains of monoclonal antibodies(6B3, 8F12 and 8G11) were stable. 【Conclusion】The three FAdV-4 penton hybridoma cell lines(6B3, 8F12 and 8G11) obtained by monoclonal antibody preparation and identification, which have good binding effect on FAdV-4 protein and have specific and broad spectrum characteristics, can steadily secrete penton monoclonal antibody. It can provide monoclonal antibody for further study on the role of penton protein in the pathogenesis of FAdV-4 infection, and lay a foundation for the development of FAdV-4 antigen detection reagents.