The invention discloses a preparation method of a lymphocyte protein sample suitable for dimensional electrophoresis, belonging to the technical field of disease pathogenesis researches. The preparation method comprises the following steps of: grinding a fresh rat spleen, filtering the ground fresh rat spleen in a 200-mesh manner, adding 4 DEG C 2ml of sodium chloride, transferring into a test tube, adding 2ml of a lymphocyte separation medium, and centrifuging to obtain a mononuclear cell; adding 2ml of double distilled water to rapidly crack RBCs (Red Blood Cells), adding 2ml of 1.8% sodium chloride to recover an isoosmotic state, washing the cells with 4 DEG C normal saline, and removing supernate; adding 2ml of 4 DEG C 5% cane sugar, mixing uniformly, and centrifuging to remove supernate; and drying a cell mass at room temperature, adding a hydration/lysis buffer to promote the dissolution of protein, ultrasonically breaking cells, and taking supernate, thus obtaining the lymphocyte protein sample suitable for the dimensional electrophoresis. With adoption of the preparation method disclosed by the invention for preparing the lymphocyte protein sample, the advantages of rapidness, simpleness and low cost are achieved, a high-quality dimensional electrophoretogram with good repeatability can be obtained, in addition, the subsequent biomass spectrometry protein analysis and identification researches are facilitated, and the preparation method can be widely applied to lymphocyte proteomics researches.