Abstract: ObjectiveMedium components suitable for in vitro culture of Paphiopedilum hirsutissimum was developed in order to provide technical support for large-scale multiplication of P. hirsutissimum by in vitro tissue culture. MethodTaking tissue culture seedlings of P. hirsutissimum as explants,basic culture medium,additives and hormone combination, which were suitable for bud induction, multiplication of cluster buds and seedling growth of P. hirsutissimum, were screened. ResultResults showed that, 1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for induction of cluster buds,with induction rate of 36.1%, and more cluster buds sprouted. 1/2MS+0.1 mg/L TDZ+3.0 mg/L 2,4-D+80.0 g/L banana juice+2.0 g/L activated carbon was optimal medium for multiplica-tion of cluster buds,the multiplication multiple was 2.02,and buds grew faster on this medium. The optimal medium for strengthening P. hirsutissimum seedling was 1.0 g/L Hyponex 1+1.0 g/L Hyponex 2+MS (organic and micro)+1.0 mg/L NAA+2.0 g/L activated carbon+80.0 g/L banana juice+1.5 g/L peptone,seedling grew well on this medium, with large biomass and rooting rate of 75.5%. ConclusionAdding banana juice to medium (1/2MS+3.0 mg/L 6-BA+0.1 mg/L NAA+2.0 g/L activated carbon) is conducive to bud induction and multiplication of P. hirsutissimum,and adding 0.1 mg/L TDZ+3.0 mg/L 2,4-D to 1/2MS+80.0 g/L banana juice has good effect on multiplication. Improved Hyponex medium is suitable for strengthening P. hirsutissimum seedling, and improved Hyponex medium supplemented with NAA and activated carbon has culture effect, but amount of added activated carbon can not be too high.