Objective To genetically subtype staphylococcal enterotoxins by applying VFDB annotation method and evaluate its accuracy.Methods VFDB annotation method and enterotoxin gene-specific PCR were applied to genetically subtype 18 staphylococcal enterotoxins （including traditional enterotoxin genes sea-see， new enterotoxin genes seg-sej and enterotoxin-like genes sek-seu） in 53 Staphylococcus aureus isolated from food in Beijing from 2009 to 2016. All the enterotoxin gene sequences obtained by VFDB annotation method were uploaded to NCBI platform and further verified by BLASTX program and refseq_protein database.Results Among the 53 Staphylococcus aureus isolates， 45 isolates （84.91%， 45/53） were identified to have one or more enterotoxin genes， and 8 isolates were without enterotoxin genes by both VFDB annotation method and gene-specific PCR. The traditional enterotoxin genes were found in 31 isolates （58.49%， 31/53）. Among the 45 isolates which carried enterotoxin genes， 16 isolates （35.56%， 16/45） had consistent enterotoxin gene typing results， and 29 isolates （64.44%， 29/45） had inconsistent results between VFDB annotation methods and PCR method. Compared with BLASTX verified results， genotypes that could be misclassified by VFDB annotation method included sea/sed， sea/sej， sea/sep， sea/ser， seg/ser and sek/sei （VFDB annotation/BLASTX verification）.Conclusion VFDB annotation could be applied as an optional method in predicting staphylococcal enterotoxin genes， but the sequence annotated as sea， seg and sek need to be further verified by BLASTX and refseq_protein database to improve the accuracy.