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 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^-1IBA, 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.