Monocye-Macrophages are key players in the immune system and dysregulation of its function will lead to uncontrollable infections, tumor and autoimmune diseases. Isolating purified monocytes from human peripheral blood is the key step for macrophages related study. Here we show a method that we used to isolate cells with the flow cell sorter by the size and granularity of cells. Sorted cells were then confirmed by staining of CD14, the surface marker of monocytes and the purity of the cells is more than 85%. This simple and efficient method can be used to isolate pure monocytes for functional research.