Abstract: Objective The aim of this study was to develop a rapid method for the Screening and identification of food-borne Clostridium perfringens isolates using multiple nucleic acid detection system combined with MALDI-TOF MS, and to provide a new strategy for the identification of C. perfringens. Methods Samples from a food poisoning incident in Zhangjiagang were collected for rapid screening with the multiplex nucleic acid detection system and MALDI-TOF MS, detection with traditional biochemical and MALDI-TOF MS, the accuracy of the results was verified by Real-time PCR combined with 16S rRNA sequencing. Results After initial screening using a multiplex nucleic acid detection system and MALDI-TOF MS, preliminary evidence indicated contamination or infection with Clostridium perfringens. Following the national standard testing protocol, 31 strains of Clostridium perfringens were identified in 122 samples associated with food poisoning incidents. Furthermore, fluorescence quantitative PCR and 16S rRNA gene sequencing were employed to verify 7 strains that showed inconsistent biochemical test results. These additional analyses ultimately confirmed that these 7 strains were indeed Clostridium perfringens. Conclusion The multiple nucleic acid detection system has the advantages of prompt detection and elimination of routine detection items, and the MALDI-TOF MS is easy to operate, rapid, has a high accuracy of detection results. The combination of the two technologies can quickly and accurately screen and confirm pathogenic factors, providing a new effective method for the detection of food poisoning pathogens.