Anoectochilus roxburghii is a wild edible species and has been traditionally used for a wide range of diseases in many countries. Our research aims to find the optimal light-emitting diode (LED) lighting conditions to improve the growth and development of A. roxburghii seedling at the acclimation stage. Two-month-old explants were cultured under the various lighting conditions including red (R), blue (B), BR (one blue: four red), BRW151 (one blue: five red: one white), BRW142 (one blue: four red: two white), and fluorescent lamp (FL). The results showed that the lighting conditions not only affect the growth and morphology of plants but also the accumulation of total flavonoids. Single wavelengths (B or R LED) inhibited the growth and secondary biosynthesis of A. roxburghii, while the BR LED showed an enhancement in both growth and biomass accumulation. A. roxburghii plants were grown under BR LED light has average plant height (7.18 cm), stem diameter (17.6mm), number of leaves (5.78 leaves/tree), leaf area (4.67 cm2), fresh weight (0.459 g/tree), dry matter percentages (11.69%), and total flavonoid (1.811 mg/g FW) is considered to be superior to FL lamps and other LEDs in the experiment. This indicates that both blue and red wavelengths are required for the normal growth of A. roxburghii. To learn more about how light affects flavonoid biosynthesis, we evaluated the expression of genes involved in this process (pal, chs, chi, and fls) and found that BR LED light enhances the expression level of chi and fls genes compared to fluorescent lamps (1.18 and 1.21 times, respectively), leading to an increase in the flavonoid content of plant. Therefore, applying BR LED during in vitro propagation of A. roxburghii could be a feasible way to improve the medicinal value of this plant.