Abstract: Utilizing the mature seeds of Dendrobium hancockii Rolfe as explants, this study investigated the impacts of various hormone combinations on the proliferation, differentiation, and rooting of protocorm-like bodies (PLBs). The results indicated that a large number of plump and green PLBs emerged from the Dendrobium hancockii Rolfe seeds after 50 days of germination cultivation. The optimal medium for proliferation and differentiation was MS（Murashige-Skoog） medium supplemented with +1.0 mg·L^–1 6-BA+0.3 mg·L^–1 NAA+30 g·L^–1 sucrose+7.8 g·L^–1 agar, with a proliferation rate of 16.67%. The optimal medium for rooting and seedling growth was MS medium + 0.5 mg·L^–1 NAA+0.1 mg·L^–1 IBA, with a rooting rate of 100% and a differentiation coefficient of 6.14, resulting in robust seedlings with rapid growth. This study optimized the tissue culture and rapid propagation system of Dendrobium hancockii Rolfe, providing a basis for further optimization and industrial production.