Abstract: Lactobacillus helveticus KLDS 1.8701, L.acidophilus KLDS AD1 and L.acidophilus KLDS AD2 were regarded as the research subject to survey their artificial gastrointestinal tract tolerance, and to evaluate the effect of them on the proliferation of mouse spleen lymphocytes in vitro, and the immunological properties of them were discussed. Results demonstrated that three lactobacillus had strong tolerance to simulated gastric and intestinal juice and bile salts, in addition, KLDS 1.8701 and KLDS AD1 were better than KLDS AD2. Three lactobacillis could accelerate the proliferation of spleen cells in a dose-dependent manner, the most obvious immunomodulation effect was observed when Lactobacillus strain suspension was at 107CFU/mL, the order of the effect of three Lactobacillus on spleen lymphocyte activity were KLDS AD1 >KLDS 1.8701>KLDS AD2. KLDS 1.8701 had the best promoting effect on spleen lymphocytes, and KLDS AD1 had promoting effect only when strain suspension was at 107CFU/mL, while KLDS AD2 had no effect.