The effects of different culture systems on the growth and kinsenoside content of Anoectochilus roxburghii (Wall.) Lindl. cultured in vitro and ex vitro were evaluated. The in vitro shoots (3 cm in height with 3 leaves) were used to investigate the effect of the in vitro hydroponic system (nylon bag (NB) and Erlenmeyer flask (EF)) with substrate (agar, cotton layer) and medium volume and with or without filter paper (FP) (Millipore membrane) on the shoot growth. After that, in vitro plantlets cultivated in ex vitro hydroponic system with substrates (cotton layer (CL) and cocopeat) combined with an alcohol burner wick on hydroponic solution yielded the biomass and kinsenoside content in the whole plant also were investigated. The results showed that a 3.000-mL EF with CL substrate (20 cm × 20 cm; 15.02 ± 0.03 g) containing 1200 mL of liquid Schenk and Hildebrandt medium (SH; Schenk and Hildebrandt, Can J Bot 50:199–204, 1972) and cover by FP resulted in better shoot growth including shoot height (7.60 cm), fresh weight (1.50 g), and dry weight (0.17 g) after 90 d of culture. Subsequent plantlet growth in a greenhouse using an ex vitro hydroponic system with CL substrate combined with an alcohol burner wick on ½ SH nutrient solution yielded the optimal results regarding plant height (14.80 cm) and fresh and dry weight (3.31 g and 0.25 g, respectively). The kinsenoside accumulation in the whole plants grown two hydroponic systems with substrate (CL and cocopeat) combining an alcohol burner wick and containing ½ SH nutrient solution yielded higher kinsenoside accumulation (1.24 to 1.32%/DW).