Objective To establish a sensitive and specific molecular biological method for detecting Staphylococcus aureus (S. aureus, SA) in laboratory animals. Methods A DNA fragment of 676 bps of the nuc gene was applified from SA genome DNA by PCR and labeled with biotin as the probe. The purified genomic DNAs of SA1800, SAm0109 and Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli were blotted onto the Hybond N nylon membrane, fixed using UV irradiation, hybridized under high stringency conditions, and detected using the Enhanced Chemiluminesence kit (ECL). Results The probe reacted positively with the SA genomic DNAs, but not with that of the non-SA with a sensitivity of I pg. 322 clinical samples taken randomly from specific pathogen free (SPF) mice were submitted to detection by the dot blotting and previously established PCR and culture with a positive detection rate of 3.1% (10/322) and an agreement of 100 %. Conclusion The dot-hybridization assay was a new rapid, sensitive and specific method and could be applied to detect SA in laboratory animals.