Abstract: 【Objective】 The objectives of this study were to optimize the aseptic spore germination and the prothallus growth conditions of Lygodium japonicum，which could provide theoretical basis for the establishment of the rapid propagation system and large-scale planting of Lygodium japonicum. 【Method】 Using Lygodium japonicum spores as experimental materials，the effects of different spore maturity levels，NaClO solution concentrations and sterilization durations，medium inorganic salt content，agar concentration，pH value of medium，inoculation concentration of spore suspension，culture temperature，dark treatment duration，and gibberellin（GA₃）concentrations were tested. Spore germination percen-tage and prothallus area were measured to identify optimal culture conditions. 【Result】 Spores from the same sporophyll exhibited varying maturity levels，with earlier mature and dispersed spores having higher germination percentage. Spores dispersed within 1-3 d achieved a final germination percentage of 38.4% after 15 d of culture，significantly higher than those dispersed later（P<0.05，the same below）. The best reproductive material was mature spores naturally dispersed within 1-3 d after sporophyll harvest. The spores treated with different NaClO solution concentrations and disinfection durations were not contaminated within 15 d of culture，but 3% and 5% high-concentration NaClO inhibited spore germination and prothallus growth. The optimal sterilization method was 1% NaClO solution soaking for 4 min. Knop’s medium with lower inorganic salt content promoted spore germination and prothallus growth. The final germination percenta-ge and prothallus area after 15 d cultured at 7.5 g/L agar concentration were significantly higher than those at 8.5 and 9.5 g/L. The final germination percentage and prothallus area spore in the medium with pH 5.8 were significantly higher than those in the media with pH 5.4 and pH 6.2. The optimal medium formula was Knop’s medium with 7.5 g/L agar，20.0 g/L sucrose，and pH value adjusted to 5.8. The optimal spore inoculation procedure was to inoculate 0.5 mL of spore suspension at a concentration of 1.0 mg/mL. The optimal culture temperature was 30 ℃ constant temperature，initiating germination on 3 d of culture and achieving a final germination percentage of 52.5% on 15 d of culture，which was higher than constant 25 ℃ and 20 ℃/30 ℃ alternating temperatures，prothallus area was also the largest. The final germination percentage of spores under dark treatment for 15 d was significantly higher than that without dark treatment（0 d），but it might not meet the efficiency requirements for large-scale reproduction. The final germination percentage of spores treated with GA₃ concentration of 0.01 mg/L was significantly higher than that without GA₃ treatment（0 mg/L），but when the GA₃ concentration reached 100.00 mg/L，the final germination percentage of spores was significantly lower than that without GA₃ treatment. 【Conclusion】 Optimal aseptic germination conditions for Lygodium japonicum spores are selected， which also favor early prothallus growth.