Objective To compare the identification effect of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, rpoB gene sequencing and VITEK 2 Compact on Staphylococci. Methods Three methods were used to identify 59 isolates of Staphylococci from food simultaneously. Results The result consistency of MALDI-TOF MS and rpoB gene sequencing was 100.00%, while the consistency of MALDI-TOF MS with VITEK 2 Compact was 83.05% (49/59). Seven isolates were misidentified by VITEK 2 Compact because of out of identification range. The MALDI-TOF MS method could quickly identify 59 strains as 17 species of Staphylococci, and the identification score was related to the species. The identification score of Staphylococcus aureus is the highest (>2.300), and the identification score of Staphylococcus squirrel, S.piscifermentans and S.succinus was relatively low (1.700-2.000). Conclusion Compared with rpoB gene sequencing and VITEK 2 Compact method, MALDI-TOF MS method is more rapid and accurate. In order to achieve a better identification effect, the existing database still needs to be further optimized.