To detect staphylococcal enterotoxins (SEs) in combination with Staphylococcus aureus (S.aureus) in an investigation of a suspected staphylococcal food poisoning (SFP) event.Methods According to epidemiological investigation, SEs were detected in the suspected food using ELISA and strains from food and patients' vomits were isolated. The presumptive isolates were identified using Vitek2 Compact and coagulase test. The identified isolates were further analyzed for the excretion of SEs and the typing of SE genes using ELISA and PCR. Pulsed-field gel electrophoresis (PFGE) was conducted for clustering analysis of the identified isolates. Results 2 and 11 S.aureus isolates were recovered from food and patients, respectively. The results of PCR and ELISA assay showed that 12 out of 13 isolates harbored enterotoxin genes of SEA, SEB, SED, SEE and expressed them simultaneously. PFGE results suggested 12 enterotoxigenic S.aureus isolates were highly homologous. Conclusion The SFP incident discussed here was caused by a S.aureus strain with unique enterotoxin phenotype. During laboratory investigation of SFP incidents, SEs detection combined with pathogen source analysis could provide reliable scientific information for such public health events.