Monoclonal antibodies (Mabs) specific for lipopolysaccharide (LPS) of Salmonella Enteritidis were evaluated in a model of LPS conjugated synthetic polymer immunization of Balb/c mice by conventional hybridoma method. Nine hybridoma cell lines were determined as antibody positive against LPS. The clone 5A8 secreting the highest antibody was selected for further characterization. Evaluated results indicate that the synthetic polymer can be used as an effective adjuvant in immunization with LPS, because the 5A8 Mab were obtained using synthetic polymer as an adjuvant. 5A8 Mab was classified as IgG2a isotype by antibody capture enzyme-linked immunosorbent assay. The reactivity of the Mab against lipid A and different LPS of Salmonella were investigated using an indirect enzyme-linked immunosorbent assay. Mab presented a wide spectrum of reactivity, coupling with antigens against Salmonella. The hybridoma 5A8 determined in this study has a great potential to be used in the development of diagnostic, prophylactic, and therapeutic agents specific for Salmonella Enteritidis and Salmonella LPS.