Abstract: To establish an efficient and stable protoplast culture system for Cibotium barometz, this study used Cibotium barometz spores as explants to systematically investigate the effects of different medium combinations on sporophyte formation. The changes in physiological and biochemical characteristics of prothallia and sporophytes at different culture stages were analyzed, and suitable materials for protoplast culture were screened for further protoplast culture studies of Cibotium barometz sporophytes. The results showed that the optimal medium for sporophyte formation was MS + 0.1 mg/L NAA + 0.2 mg/L KT. The TP content, as well as the activities of POD and SOD, were significantly higher in sporophytes than in prothalli. Using sporophyte protoplasts as the culture material, the best culture results were achieved in a shallow liquid medium of KM8P + 1.0 mg/L 6-BA + 0.5 mg/L 2,4-D + 6.0% glucose + 2.0% sucrose, with a density of 1×10⁵ cells/mL. Under these conditions, the first division of protoplasts occurred at 2 days, small cell clusters were observed at 14 days, and callus tissues of approximately 2 mm in size developed after 28 days. The callus tissues could continuously proliferate on MS solid medium. This study can provide key technical parameters for the protoplast culture and plant regeneration system of Cibotium barometz.