In this study, an immunology-based assay that employed specific monoclonal antibodies binding with somatic or flagella antigens of Salmonella enterica subsp. enterica was performed. As this pathogen is one of the most important bacterial species responsible for foodborne outbreaks, its detection in food by rapid and easy methods is properly suitable. After a first screening by indirect ELISA, three monoclonal antibodies (1B6D9, 1B6C11, 1D12F11) versusS. enterica subsp. enterica serovar Typhimurium ATCC 14028 (whole antigen) and another one (4E6F11) versusS. enterica flagellin were further characterized by immunoblotting and mass spectrometry analysis. Then, a total of 84 food samples (dairy products, meat, pasta and flour, eggs, and animal feed) were analyzed by both the official method ISO 6579:2002 and S. enterica capture ELISA. For the standardization of the last method, the specific monoclonal antibody 4E6F11 was selected. The developed Salmonella capture ELISA showed a significant agreement with the official method (ISO 6579:2002). Relative sensitivity, specificity, and accuracy were 100%, 81.0%, and 90.5%, respectively. Therefore, this assay could represent a valid alternative to conventional methods able to detect this pathogen in food.