Background <p><i>Salmonella</i> is an important zoonotic pathogen that is widely distributed in every link between livestock and poultry production. <i>Salmonella</i> contamination not only affects the healthy development of the livestock industry but also poses a serious threat to public health, as it can infect humans through farming, processing, and consumption of meat and egg products. Therefore, establishing detection methods capable of simultaneously identifying different <i>Salmonella</i> serotypes is crucial.</p> Results <p>This study focused on using <i>Salmonella</i> flagellin as an immunogen to screen and prepare monoclonal antibodies, ultimately establishing a blocking ELISA (bELISA). In this study, flagellin from <i>S.</i> Enteritidis was extracted by acid lysis and used to immunize BALB/c mice. Cell fusion was performed following three immunizations, resulting in the generation of six hybridoma cell lines that stably secreted monoclonal antibodies. Monoclonal antibody (MAb) of 1F6 exhibited excellent reactivity with <i>Salmonella</i> flagellin, demonstrating an affinity of 1.11 × 10¹⁰&#xa0;M⁻¹. Specificity analysis indicated that MAb 1F6 reacted with all flagellated <i>Salmonella</i> species, but not with non-flagellated <i>S.</i> Pullorum or other bacterial species, confirming its strong specificity and suitability for this method. Subsequently, an array titration method was employed to determine the optimal coating concentration of the antigen for the bELISA, which was found to be 1&#xa0;μg/ml, while the optimal concentration for the enzyme-labeled antibody was 534&#xa0;ng/ml. Various experimental optimizations were conducted to identify the optimal detection conditions. The results demonstrated specific recognition of <i>Salmonella</i> and no cross-reactivity with other sera, when testing chicken sera infected with eleven different pathogenic microorganisms. Furthermore, this method was applied to 227 chicken serum samples, achieving a conformity rate of 97.35%. In addition, 205 serum samples from layer chickens at a certain farm were analyzed, and <i>Salmonella</i> was detected on day 90, with an overall positive rate of 4%, consistent with previous studies, indicating that our method can be used for <i>Salmonella</i> monitoring in production enterprises.</p> Conclusions <p>The bELISA method was developed by utilizing <i>Salmonella</i> flagellin as an antigen and exhibited excellent stability, strong specificity, and sensitivity, making it suitable for broad-spectrum screening of <i>Salmonella</i> in infected chicken.</p>