The invention discloses a flow cytometry detection method for mouse thymus or spleen T-lymphocyte subsets. The method comprises the following steps: acquiring thymus or spleen tissues of tested mice, and preparing single-cell suspension; respectively sucking the single-cell suspension of the thymus or spleen, respectively adding anti-mouse DC3, CD4 and CD8 monoclonal antibodies, swirling and uniformly mixing, keeping away from light at the temperature of 4 DEG C and staining; washing with PBS (Phosphate Buffer Solution) after staining, resuspending the cells, and detecting by using flow cytometry; and analyzing the detection result, thereby obtaining the percentage of each T-lymphocyte subset of the thymus or spleen of the mice. The stable and standardized method for detecting the mouse thymus or spleen T-lymphocyte subsets through flow cytometry is successfully established in the invention. By improving the problems such as target cell selection, compensative regulation and parameter selection in the sample pretreatment, detection and analysis process, errors caused by human factors in the test process are avoided, and the authenticity and objectivity of result judgment are improved.