[Objective] The aim was to study the conditions of tissue culture regeneration seedling by using the stem segments of Euphorbia tirucalli and determine the optimum culture condition of each culture stage,so as to provide references for the factory production and relative study of tissue culture seedling of E. tirucalli. [Method] Taking the stem segments of E. tirucalli as explants,the effects of various mediums on germination rate,multiplication coefficient and rooting rate were studied. [Result] The optimum induction medium of germination culture was 1/2MS+NAA 0.02 mg/L+6-BA 1.0 mg/L,with differentiation rate of 89.7%; the best subculture medium was 1/2MS+NAA 0.02 mg/L+6-BA 0.60 mg/L+AD 3.0 mg/L,with multiplication coefficient of 5.70; the optimum rooting culture medium was 1/2MS+NAA 0.40 mg/L+IBA 0.4 mg/L,with rooting rate of 100% and transplanting survival rate of 80%. [Conclusion] The tissue culture conditions of stem segments of E. tirucalli were determined primarily.