Abstract: Conditions of tissue culture for Anoectochilus roxburghii were optimized to maximize the content of effective substances in the plantlets. The tissues were cultivated in test tubes containing a basal medium of MS+6-BA 2.0 mg·L^-1+NAA 0.2 mg·L^-1+KT 1.5 mg·L^-1+agar 7 g·L^-1+glucose 25 g·L^-1. Effects of various organic additives, activated carbon additions and light exposures on the accumulation of polysaccharides and total flavonoids during the growth periods of the plantlets were studied using a L9 (3⁴) orthogonal experimental design. It was found that the treatment effects were in the order of:light > additives > activated carbon. The average increases on the target substances by the blue light exposure were significantly higher than those by the white or red light (P < 0.05). The increases by adding banana mud to the basal medium were significantly greater than the addition of either apple juice or carrot juice (P < 0.05). And, the gains by inclusion of 1.0 g·L^-1 of activated carbon in the medium were significantly higher than those at 1.5 g·L^-1 level (P < 0.05). Thus, it was concluded that an optimal production of polysaccharides and flavonoids in the plantlets could be achieved by a combination of the following ingredients in the medium under blue light:MS+6-BA 2.0 mg·L^-1+NAA 0.2 mg·L^-1+ KT1.5 mg·L^-1+agar 7 g·L^-1+glucose 25 g·L^-1+banana puree 100 g·L^-1+activated carbon 1.0 g·L^-1.