Abstract: In order to detect the Acinetobacter lwoffii quickly and accurately,a real-time PCR detection based on SYBR GreenⅠ was established with a pair of primers designed according to the β-lactamase gene of A.lwoffii( AL-bla_(OXA-134)).The result showed that this method could detect the A.lwoffii quickly in raw milk with a detection limit of 2.4 × 10~2 cfu/m L,and without any cross-reaction to other microorganisms in raw milk. The standard curve was y =-3.838 x + 40.93,of which correlation coefficient was 0.993. The detection limit of A. lwoffii in artificial contaminated milk sample was 2.4 × 10~3 cfu/m L by this method.Finally,milk sample collected from eight pastures in lower reaches of the Yangtze River since Nov.,2015 to Jan.,2016 was analyzed by this method.The method established in this study was specific,highly sensitive,which could be further used in detection of A.lwoffii in raw milk and helpful to improve the quality safety of raw milk.