The rapid identification of Salmonella, a significant foodborne pathogen, is of significant importance. To establish a new method for identifying Salmonella based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), this study optimized culture medium type, incubation time, bacterial lysis methods, and instrumental parameters. The results showed that compared to the national standard method GB/T 33682-2025 General microorganism identification method with matrix-assisted laser desorption/ionization time of flight mass spectrometry, the recovery rate increased from 54.7% to 84.7% after replacing the culture medium with nutrient agar. Increasing the incubation time from 24 to 48 h increased the average identification score by 7.0%, from 2.203 to 2.355. Optimizing lysis methods elevated the average identification score by 19.0%, from 1.898 to 2.259. When the instrumental parameters were adjusted to 50% laser energy and a laser wavelength of 370 nm, the average identification score increased to 2.211. Compared to its original version specified in GB/T 33682-2025, the established MALDI-TOF/TOF MS method exhibited improved accuracy and reliability in Salmonella identification.