Effects of different disinfection treatments, additives, plant growth regulators on tissue culture and rapid propagation system of Habenaria rhodocheila with seeds as explants were studied. Sterilizing the seeds with 0.1% mercuric chloride for 15 min was the best disinfection treatment. Seeds germinated only on the medium adding active carbon(AC). 0.2% active carbon in culture medium was fit for germination. The optimal medium for seed germination was 1/2MS＋NAA 0.5 mg·L-1＋AC 0.2%, the optimal medium for multiple buds formation was 1/2MS＋NAA 0.2 mg·L-1＋ZT 3.0 mg·L-1＋AC 0.2%, the optimal rooting medium was 1/2MS＋NAA 0.1 mg·L-1＋6-BA 2.0 mg·L-1＋AC 0.2%.