To optimize tissue culture techniques of Alsophila spinulosa to improve the success rate and productivity of tissue culture. A.spinulosa spores were used as explants to study the effects on the four processes: spore-induced gametophytes by different growth regulators, prothallus green globular body (PGGB) induction, PGGB differentiation and sporophyte rooting. Its was studied to further explore the survival effects of different substrates on the transplantation of A.spinulosa tissue culture seedlings. The results show that：(1)Spore-induced gametophytes: The best induction effect was 1/8MS+0.5 mg/L 6-BA+0.1 mg/L NAA+sucrose 20 g/L+agar 10 g/L, with an induction rate of 64.6%and induction days of 23 d. (2)PGGB induction：The induction rate of 1/2MS+0.3 mg/L 6-BA0.3 mg/L+0.4 mg/L NAA+sucrose 30 g/L+agar 6 g/L reached 100%, the fresh weight of PGGB was 1.13 g, the multiplicationratio was 11.04 and the diameter was 2.01 cm. (3)PGGB differentiation：The best differentiation effect was obtained at 1/2MS+0.3 mg/L KT+0.4 mg/L IBA +sucrose 30 g/L+agar 6 g/L, with a differentiation rate of 92.1% and a PGGB average of 41.67 differentiated buds. (4)sporophyte rooting：The rooting rate of spores in 1/2MS+1.0 mg/L IBA+sucrose 30 g/L+agar 6 g/L was 100%. (5)Acclimatization：The survival rate of tissue culture seedlings transplanted in the substrate (red soil: humus soil: peat soil=2:1:1) was 96%. The establishment of A.spinulosa PGGB induction and regeneration system is affected by plant growth regulators, and the survival rate of tissue culture seedlings is significantly different. This study successfully constructed an efficient reproduction and regeneration system of A.spinulosa, providing technical support for furtherexploring the development mechanism of A.spinulosa in vitro culture, new species, large-scale production, and application of A.spinulosa in soil matrix.