Abstract: In order to raise efficiency of planting seedling by artificial spore culture of Athyrium multidentatum, in this article, spore of Athyrium multidentatum was taken as the explants, and used different categories of hormone, NaH2PO4 and AC during the period of tissue culture. The result showed that the methods of plant tissue culture were used for effects 5 levels of time of treatment of GA3,4 hormone(NAA, 2,4-D, BA, KT) of 5 levels concentration, 2 cultural ground substances,5 levels of GA3 concentration applying on spores germination; the effects 6 levels NaH2PO4 and 5 levels AC on planting rate were studied. The results showed that the 20mg/LGA3 most suitable treatment time was 5 min, the most suitable hormone was KT, BA and NAA take second place, the most suitable medium was 1/2MS on spores germination; The most suitable concentration of NaH2PO4 was 200~300mg/L, the most suitable AC was 2% in cultural ground substances on planting rate.