Abstract: The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) diagnostic tool was used to rapidly detect and identify three foodborne pathogens, Salmonella, Staphylococcus aureus and Bacillus cereus in ready-to-eat bulk foods in this study. The best method was identified by optimizing and screening various experimental conditions such as spot sampling method, pre-treatment method, different media and culture time, and its detection limit and stability were evaluated. Meanwhile, the established method was compared with multiplex PCR and the other biochemical identification methods such as VITEK 2 to contrast the consistency of the three identification results. Finally, batch food sample testing was carried out to explore the applicability of using MALDI-TOF MS to test ready-to-eat bulk foods. Results indicated that all the identification scores of the three bacteria were higher than 95% for the same batch and different passages, with a coefficient of variation of less than 2%. It demonstrated that the established method has good repeatability and stability, and the detection limit was 10⁵ CFU/mL. The results of strain identification by MALDI-TOF MS were highly consistent with those of multiplex PCR and biochemical identification methods. Moreover, the MALDI-TOF MS method was more specific and efficient. Regarding spot sampling, the three covering methods showed higher average identification scores than the sandwich and mixed methods, all of which scored 99.9%. The 1 μL+1 μL covering method spot sampling approach obtained higher average peak numbers, and more complex and stronger characteristic peaks. Regarding pre-treatment, the formic-acid-acetonitrile method obtained a higher peak volume and peak intensity than other bacterial treatment methods, with an average identification score of 98% or higher. At the same time, the matching degree with the database spectrum was higher. In summary, MALDI-TOF MS method was not only sensitive, specific, accurate and rapid in foodborn pathogen identification, but also had simple operation and intuitive in results. As a powerful supplement to the national standard method, it provided a viable alternative for analysing, identifying and typing foodborne pathogens.