With tender stem segments of Symplocos paniculata as the experiment material, the tissue culture system of S. paniculata was preliminarily established to research the key factors influencing initiation culture, proliferation, rooting culture and transplantation. Treatment with 0.1% HgCl₂ for 3 min was the optimum scheme for explant sterilization of S. paniculata; survival rate of sterile seedlings was 81%. The rate of germination of initiation culture was 86.83% in the medium with 1/2MS+30 g∙L^-1 sucrose+8 g∙L^-1 agar. The proliferation coefficient was 3.57 in optimum culture medium with 1/2MS+1.0 mg∙L^-1 6-BA+0.02 mg∙L^-1 IBA+30 g∙L^-1 sucrose+8 g∙L^-1 agar. The suitable rooting medium was WPM+0.5 mg∙L^-1 IBA+0.5 mg∙L^-1 NAA+20 g∙L^-1 sucrose+2 g∙L^-1 AC+8 g∙L^-1 agar, and rooting rate reached 93%. After being acclimatized, the regenerated plants were transplanted into substrate of garden soil and peat soil (v:v=1:1), with survival rate of regenerated plants of 83%.