Abstract: It was studied on four factors of basic medium, 6-BA, NAA and sucrose on adventitious buds proliferation culture and rooting culture to optimize the rapid propagation medium of Anoectochilus formosanus by orthogonal design L9(34), taking the apical buds of test-tube seeding as the explants. The results showed that: four factors had a very significant impact on the adventitious buds proliferation culture and rooting culture. The effect for proliferation culture: 6-BA> sucrose> NAA> the basic medium, the effect for rooting culture: sucrose> 6-BA> the basic medium> NAA. The optimizing medium for proliferation culture was MS+6-BA 3.0 mg/L+NAA 0.3 mg/L+ sucrose 20 g/L. The optimizing medium for rooting culture was 1/2MS+ 6-BA 1.0 mg/L+ NAA 0.6 mg/L+ sucrose 40 g/L.