Abstract: To establish rapid separation method for food-borne Staphylococcus aureus (SA) based on immunomagnetic separation by test optimizing, it provided a basis for follow-up rapid detection.The immunomagnetic nanobeads (IMBs) were used to capture the S.aureus.By calculating the capture efficiency combining with the colony counting, the amount of the SA polyclonal antibody (4⁵mg/mL) coating the magnetic beads, the amount of IMBs and response time were investigated for influence on capture efficiency.The orthogonal test was done based on the single factors test.It was preliminarily studied on the sensitivity, specificity and the practical application of this method in food sample.The results showed that the optimal condition for capture were the amount of antibody 30μL, the amount of IMBs 80μL, and response time 45min, respectively.Under the optimized conditions, the capture efficiency was above 30%.The capture limit could reach 101CFU/mL of SA and its specificity was good.The method was used to the rapid separation of SA in lamb wash water at less than 1h capture time.Taken together, the immunomagnetic separation, as one of simple rapid separation and screening methods, could be applied to rapidly separate food-borne pathogens, such as S.aureus.