Abstract: To develop a duplex PCR assay for simultaneous detections of Staphylococcus aureus and Bordetella bronchiseptica in rabbits, two pairs of specific primers were designed based on the conserved regions of nuc and fimN genes of the pathogens. The methodology with optimized reaction conditions was specific and free of cross-reactions with Pasteurella multocida, Escherichia coli and Clostridieum welchii. The detection limit of the method was 5 pg genome DNA on both pathogens. Moreover, the results obtained by using the duplex PCR assay were in perfect agreement with those determined separately by single PCR determinations. Thus, the newly developed highly specific, sensitive and accurate methodology could be a rapid assay applied for simultaneous identification of S. aureus and B. bronchiseptica present in a diseased rabbit.