Abstract: Pick-off blood with anticoagulant aseptically and the lymphocytes were isolated. Effects of FCS，PBMC，PHA-P of different concentration on promote lymphocytes proliferation were tested by orthogonal test. On the base of the best level combination, different concentration pig TF was investigated to detect its effect on the activation of lymphocytes by MTT essay. The result of experiment demonstrate that the best levels combination of canine lymphocyte，PHA-P, calf blood serum were 2.5×106 unit/ml，6.25 μg/ml，10% respectively. Under this optimum cultural condition, the lymphocytes remarkable proliferated between 0.195~25mg/ml of the pig TF，and the best stimulate concentration of the pig TF only or it with PHA-P is 1.56mg/mL. Furthermore, it is confirmed that lymphocytes developed by the pig TF single was much better than it cooperated with PHA-P. The results confirm that the difficult race TF’s effect was obvious and provides a basis for further application on the difficult race TF preventing and curing canine’s virus disease.