Objective The aim of this study is to separate,cultivate and identify mouse peripheral blood monocyte/macrophages( PBM) in L929- conditioned medium. Methods Mouse PBM was isolated by centrifugation,and cultured with L929-conditioned medium for 7 days. PBM was identified by immunofluorescence and flow cytometry( FCM). Results PBM display the immunophenotype of monocyte / macrophage and with phagocytic function. Conclusion It was a simple method for separation,culture and identification of mouse PBM in vitro.