The effects of different light conditions on the flowering of Dendrobium officinale plantlets in vitro were determined. Single nodes of D. officinale (approx. 2.0 cm long with 2-3 leaves) were grown for 100 days under six different light conditions created by light emitting diodes. The six light conditions were as follows: blue and red at 660 nm or 630 nm (R⁶⁶⁰B and R₆₃₀B), blue and green and red (R₆₆₀BG and R₆₃₀BG), and blue and yellow and red (R₆₆₀BY and R₆₃₀BY). Cultures grown under fluorescent lamps (FL) served as a control. The indices of growth, flowering, and floral development of D. officinale plantlets in vitro were investigated. The results showed that the 630-nm red light resulted in greater plant height and earlier flowering. Compared with 630-nm red light, 660-nm red light in the complex spectrum resulted in greater plantlet and flower dry mass, larger stem diameter, and peduncles, thicker leaves and petals, a longer blooming period, a greater number of open flowers, higher flowering percentage, increased pollen vigor, and higher carotenoid contents in petals. Green light added to the complex spectra of red and blue light resulted in greater plantlet dry mass, larger stem diameter, and thicker leaves. Yellow light promoted carotenoid accumulation in the petals. Red light at 660-nm promoted sugar transport from leaves to stems and stem tips, thereby promoting flowering.