Abstract: In order to establish a tissue culture and rapid propagation system for in vitro and out of vitro seedling formation of Asplenium nidus, the factors affecting in vitro propagation and seedling formation of A. nidus were studied using mature spores of A. nidus as experimental materials. The results showed that the optimal medium for spore germination was 1/4 MS, and the germination rate reached 91.75%, while the gametophytes proliferated best in the medium of 1/2 MS+6-BA 0.3 mg·L^-1+NAA 0.2 mg·L^-1, and the average proliferation coefficient was 5.1. The addition of sterile water or liquid 1/2 MS to the medium was helpful for the formation of seedlings in vitro. After that, GGB induction rate was the highest in MS+6-BA 0.5 mg·L^-1+NAA 0.2 mg·L^-1 medium, reaching 94.33%, which realized the rapid proliferation by GGB pathway. The seedlings induced by GGB rooting best in 1/2 MS medium with IBA 1 mg·L^-1, the rooting rate reached 100%. In the test of in vitro seedling formation, spraying 1 g·L^-1 KH₂PO₄ solution could promote the seedling formation of gametophytes, and the seedling formation rate reached 82%.