Abstract: Polystichum caruifolium is a fern endemic to China, with scarce wild resources and slow natural regeneration. In order to establish its artificial rapid propagation system, taking its mature spores as materials, research on tissue culture and rapid propagation regarding spore germination, prothallium proliferation and the GGB pathway of prothallium was carried out. The results showed that the optimal medium for spore germination of Polystichum caruifolium was 1/4MS. The spores sown in this medium germinated earliest, 1 to 7 days earlier than those under other treatments. The germination was neat and the germination rate was the highest, reaching 70.3%. Moreover, the spores grew rapidly after germination. Both exogenous hormones NAA and 6-BA could induce the proliferation of prothallium, and the proliferation multiples were mostly above 4.5. The optimal medium formulations for the best prothallium proliferation effect were 1/2MS + 6-BA 1.0 mg/L or 1/2MS + NAA 0.3 mg/L, with the proliferation multiples reaching 5.2 times and 5 times respectively. The interaction between hormones NAA and 6-BA could induce the prothallium to develop into green globular bodies (GGB). The optimal medium for inducing GGB from prothallium was 1/2MS + 6-BA 1.0 mg/L + NAA 0.3 mg/L. The seedling formation pathway through proliferated prothallium was superior to that through inducing GGB from prothallium, because the former required less time for seedling formation and had better seedling quality. From the analysis of the influence of culture conditions on seedling formation, exogenous hormones NAA and 6-BA inhibited the differentiation of sporophyte seedlings. Within a certain range, a proper increase in the concentration of inorganic salts could promote the formation of young sporophytes. Considering the seedling formation time, the amount of seedlings formed and the growth of small seedlings, the optimal medium for the differentiation of GGB into seedlings was MS, while the optimal medium for the induction of prothallium into seedlings was 1/2MS, and the highest seedling formation rate could reach 70.8%.