In this study, micropropagation and hairy root culture of Ophiorrhiza alata for camptothecin production were investigated. Leaf and node explants from germinated seed of O. alata were cultured on half strength Murashige & Skoog (MS) medium variously supplemented with Kinetin (Kn) and α- naphthaleneacetic acid (NAA) for shoots multiplication. Multiple shoot explants were induced roots and hairy roots by indole-3-butyric acid (IBA) and Agrobacterium rhizogenes TISTR 1450, respectively. Abundant hairy roots were developed at the wound sites. Survival rates between root plantlet induced by plant growth regulator and root plantlet induced by A. rhizogenes were 100 and 80 %, respectively when transplanting to greenhouse. Various O. alata plant types from growing in soil, in vitro culture, hairy root culture, and transplanting to greenhouse were analyzed and compared the amount of camptothecin content by High Performance Liquid Chromatography. Untransformed root of soil–grown plants had the highest amount of CPT 39.98 µg/g dry wt. Leaf from soil–grown plant had the level of CPT closely to in vitro culture including root and hairy root induction with approximately 10 µg/g dry wt. while leaf from in vitro culture had CPT content only 6.29 µg/g dry wt. Four week after transplanting to greenhouse, both root and leaf from induced for rooting by IBA plant and composite plant had CPT content approximately 3 µg/g dry wt. These results indicated that in vitro culture and hairy root culture of O. alata had potential for campothecin production. Even though the camptothecin content in hairy root was lower than roots from soil grown plant, hairy root could produce the biomass accumulation and it is not destruction for nature plant.