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<i>Staphylococcus aureus</i> is a common food pathogen and has a strong tolerance to environmental stress. Here, the biofilm formation of <i>S. aureus</i> strains after cold stress for 24 weeks were investigated. It was found that the biofilm formation of <i>S. aureus</i> CICC 21600, CICC 22942, W1, W3, and C1 cells was enhanced after cold stress for 20 weeks. What is more, the mRNA levels of the <i>clfA</i>, <i>icaA</i>, <i>icaB</i>, <i>icaC</i> or <i>icaD</i> genes in these strains were increased for >2-fold. The increased gene transcription levels were consistent with the increase in the polysaccharide content in the biofilm matrix of these <i>S. aureus</i> strains after cold stress. Meanwhile, hydrophobicity and the adhesion proteins also played a role in the formation of biofilms. The biofilm of <i>S. aureus</i> cells can be effectively degraded by snailase and proteinase K (125 µg/mL + 20 µg/mL) mixture. In summary, <i>S. aureus</i> frozen at −20 °C for 12 to 20 weeks is still a potential hazard. Food factory equipment should be cleaned in a timely manner to avoid outbreaks of foodborne pathogenic bacteria due to contamination.