Abstract: In order to promote the protection and utilization of the resources of Stephania tetrandra S.Moore, a plant tissue culture system was established using stem segments and callus tissue as materials, and suspension culture was carried out using callus tissue cells. Result showed that the optimal culture medium for direct differentiation of adventitious buds from stem segments was MS+6-BA 1.5 mg/L+NAA 0.2 mg/L. The induction rate of adventitious buds was 100% after 60 days, with 16 adventitious buds differentiated from a single explant and up to 28 at 90 days. This formula was also suitable for inducing callus tissue differentiation of adventitious buds, MS+IAA 0.2 mg/L was the best method for adventitious bud rooting, with a rooting rate of 92.5% after 30 days, an average main root length of 7.78 cm, and a survival rate of 88.00% after 15 days of seedling cultivation and transplantation. Suspension culture using callus cells could achieve significant bioaccumulation in a shorter culture cycle, and the qualitative detection of alkaloids in dry culture extract was positive.