Adiantum nelumboides is a rare and endangered fern that is only distributed in China’s Three Gorges Reservoir area. Its wild population has decreased, and it has been listed as the national first-level essential protection plant. Propagating by shoot division or spore is the primary propagation method of Adiantum nelumboides, and spore propagation is more advantageous because of its high reproductive efficiency. To provide the technical basis for ex-situ conservation, it is necessary to standardize the environmental factors required for spore reproduction and determine the spore culture conditions with the highest reproductive efficiency. To understand the effects of various factors on the culture of Adiantum nelumboides, we adopt spore culture to study the effects of substrate, temperature, illumination intensity, sowing density, spore storage time, and spore disinfection, which can provide a scientific basis for conservation and resource utilization. The results showed that spore germination needs light. The most suitable environmental conditions for spore germination, gametophyte development, and young saprophyte growth were as follows: the substrate was peat: vermiculite=1:1, the temperature was 25℃, the illumination intensity of fluorescent lamp was 3 000 lux, sowing density was 25 pcs/cm2, spore storage time was 1 m and not need to be disinfected. This study provides the best environmental conditions for the spore culture of Adiantum nelumboides. Through spore propagation, many sexual reproduction individuals can be obtained, which provides technical standards for the conservation of Adiantum nelumboides.