The present disclosure provides a device for separating cells in fluid, comprising a first driving pump, a separation column, a detection column, a second driving pump, and several three-way valves and secondary driving pumps; one end of the separation column is connected with the first driving pump through a first three-way valve; the other end is connected with the second driving pump through a second three-way valve; the separation column includes at least five sub filtration columns in parallel; the sub filtration column comprises a fixing bracket and a track-etched membrane of polycarbonate or polyester material attached to the bottom of and the side surfaces all around the fixing bracket, wherein the pore diameter of the track-etched membrane is 5-25 μm. The device of the present disclosure not only provides a new method for accurately determining whether there are circulating tumor cells in the blood of the living animal and provides a new method for sorting and counting the circulating tumor cells in the blood of the living animal, but also provides a pioneering new method and new device for therapy of the tumor transfer and removal of the tumor cells inside the body, and has extremely high economic and social values.