AbstractStaphylococcus aureus (S. aureus) is a common zoonotic foodborne pathogen that poses a serious threat to the microbial control of meat processing, especially the multidrug resistant (MDR) strain. This study aimed to investigate the prevalence, antimicrobial susceptibility, virulence genes, biofilm formation ability (BFA), and molecular types of S. aureus isolated from two beef‐processing plants in China. The results revealed that the prevalence of S. aureus was 17.1% (60/350), including one methicillin‐resistant strain. The chilled carcass showed the highest detection rate among the seven sampling points. The isolates exhibited high resistance to penicillin (88.3%) and lincomycin (80.0%), and 75.0% of the isolates showed multidrug resistance (MDR). All isolates carried hla and clfa and critical toxin genes (seb, pvl, and tst) were also detected. Additionally, 96.7% of isolates exhibited strong and moderate BFA. The isolates were typed by Multilocus Sequence Typing (MLST) and Staphylococcus Protein A (spa) typing methods, resulting in nine different types for each method. The predominant type was ST7‐t091, accounting for 68.3% of the isolates. Our research suggests that there may be a critical control point in the chilling room, and the high MDR rate and strong BFA increase the difficulty of control and the risk of transmission.