Staphylococcus aureus, a commensal and a pathogenic bacterium, causes a wide variety of diseases in humans and animals with a high impact on public health and the livestock industry. The risk of zoonotic transmission to humans highlights the need to understand the molecular ecology of S. aureus in foods. In this study, we obtained 25 S. aureus isolates from 39 crayfish samples in Hubei, China. PCR was applied for detection of presence of virulence and methicillin resistance genes in the pathogen genome. The result revealed that all of the 25 S. aureus isolates harbored at least four virulence genes, and 64 % of them were positive for five or more virulence genes. The most predominant virulence genes were coa, alpha-HL and beta-HL genes (100 %), followed by sea (68 %), fnbA (60 %), tsst-1 (36 %), while none of the examined isolates presented positive for mecA gene conferring methicillin resistance. Subsequently, all of the isolates were assessed for phenotypic biofilm formation with the microtiter plate assay. The results showed 92 % isolates could produce biofilm with different forming capacity. Multilocus sequence typing divided the isolates into five sequence types (STs), three of which (ST1920, ST188 and ST398) were the same with the isolates from livestock and clinic in China. This study provides preliminary insights into the genetic diversity and virulence gene profiles of S. aureus from crayfish, suggesting that S. aureus isolates from crayfish is a potential hazard for consumers and deserves further attention.