Salmonella enterica subsp. enterica serotype Typhimurium is one of the major causative agents of human gastroenteritis. Here we raised a panel of 45 monoclonal antibodies (MAbs) against ser. Typhimurium DT104 by immunizing mice with formalin-killed bacteria and demonstrated that all the MAbs recognized the bacterial lipopolysaccharide (LPS) antigen. These MAbs were specific for group O:4 Salmonella with very little or no cross-reactivity with other closely related bacteria and were able to bind to the cell surface of live bacterial cells, making them potential candidates for capture and concentration of the pathogen in food and water samples. Epitope characterization revealed that the O:5 antigen present in the LPS of some serogroup 4 Salmonella is the critical factor for the binding of these MAbs to LPS. This study has provided some insights into the structure of the Salmonella LPS and its influence on the antigenicity of LPS.