A method for preparing flow cytometric sample in human immune cells in vitro for flow cytometry, characterized by comprising the steps of: 1) separating plasma and blood precipitation by centrifuging in vitro anticoagulant blood samples to obtain blood cell precipitate; 2) mixing the blood cell precipitate with PBS solution to obtain a blood cell dilution; 3) adding the blood cell dilution into a centrifuge tube containing the lymphocyte separation solution having a volume equal to that of the blood cell dilution, centrifuging, and discarding plasma to obtain buffy coat cells; 4) adding the buffy coat cells to a centrifuge tube, adding PBS solution under stirring, centrifuging, discarding supernatant, and then adding PBS solution to adjust the cell concentration to provide lymphocyte suspension having a concentration of 1×10 5 /ml - 1×10 8 /ml, further preferably 1.5×10 6 /ml - 3×10 6 /ml; 5) adding the lymphocyte suspension into a flow tube, then adding flow cytometric antibody to label lymphocyte subpopulations, mixing evenly, and incubating at 2-8°C without light; 6) adding PBS solution to the flow tube, mixing, and centrifuging to remove unbound antibody; 7) completion of samples; 1 if a detection on a flow cytometer is performed immediately, the supernatant is discarded and the precipitate is mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection; or 2 if the detection cannot be performed immediately, formalin is added, mixed, and left to stand at 2-8°Cwithout light; and before detection, each of the tubes is added with PBS solution, centrifuged to discard the supernatant, and evenly mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection. 1) separating plasma and blood precipitation by centrifuging in vitro anticoagulant blood samples to obtain blood cell precipitate; 2) mixing the blood cell precipitate with PBS solution to obtain a blood cell dilution; 3) adding the blood cell dilution into a centrifuge tube containing the lymphocyte separation solution having a volume equal to that of the blood cell dilution, centrifuging, and discarding plasma to obtain buffy coat cells; 4) adding the buffy coat cells to a centrifuge tube, adding PBS solution under stirring, centrifuging, discarding supernatant, and then adding PBS solution to adjust the cell concentration to provide lymphocyte suspension having a concentration of 1×10 5 /ml - 1×10 8 /ml, further preferably 1.5×10 6 /ml - 3×10 6 /ml; 5) adding the lymphocyte suspension into a flow tube, then adding flow cytometric antibody to label lymphocyte subpopulations, mixing evenly, and incubating at 2-8°C without light; 6) adding PBS solution to the flow tube, mixing, and centrifuging to remove unbound antibody; 7) completion of samples; 1 if a detection on a flow cytometer is performed immediately, the supernatant is discarded and the precipitate is mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection; or 2 if the detection cannot be performed immediately, formalin is added, mixed, and left to stand at 2-8°Cwithout light; and before detection, each of the tubes is added with PBS solution, centrifuged to discard the supernatant, and evenly mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection. 1 if a detection on a flow cytometer is performed immediately, the supernatant is discarded and the precipitate is mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection; or 2 if the detection cannot be performed immediately, formalin is added, mixed, and left to stand at 2-8°Cwithout light; and before detection, each of the tubes is added with PBS solution, centrifuged to discard the supernatant, and evenly mixed with PBS-EDTA solution at 2-8°C for flow cytometric detection.