Light spectrum plays an important role in regulating the growth and development of in vitro cultured potato (Solanum tuberosum L.) plantlets. The status of potato plantlets at the end of in vitro stage influences the minituber production after transplanting. With 100 μmol m–2 s–1 total photosynthetic photon flux density (PPFD), a light spectrum study of 100％ red light emitting diodes (LEDs) light spectrum (RR), 100％ blue LEDs light spectrum (BB), 65％ red+35％ blue LEDs light spectrum (RB), and 45％ red+35％ blue+20％ green LEDs light spectrum (RBG) providing illumination at the in vitro cultured stage of potato plantlets for 4 weeks using fluorescent lamp as control (CK) was performed to investigate the effects of LEDs light spectrum on the growth, leaf anatomy, and chloroplast ultrastructure of potato plantlets in vitro as well as the minituber yield after 2 months transplanting in the greenhouse. Compared to CK, RB and RBG promoted the growth of potato plantlets in vitro with increased stem diameter, plantlet fresh weight, plantlet dry weight, and health index. Furthermore, BB induced the greatest stem diameter as well as the highest health index in potato plantlets in vitro. Root activity, soluble protein, and free amino acid were also significantly enhanced by BB, whereas carbohydrates were improved by RR. In addition, thickness of leaf, palisade parenchyma and spongy parenchyma was significantly increased by BB and RBG. Chloroplasts under BB and RBG showed well-developed grana thylakoid and stroma thylakoid. Unexpectedly, distinct upper epidermis with greatest thickness was induced and palisade parenchyma and spongy parenchyma were arranged neatly in RR. After transplanting in the greenhouse for 2 months, potato plantlets in vitro from BB, RB, and RBG produced high percentage of large size tuber. BB improved fresh and dry weights of the biggest tuber but decreased tuber number per plantlet. In addition, RBG increased tuber number as well as tuber fresh and dry weight slightly. Our results suggested monochromatic blue LEDs as well as combined red, blue or/and green LEDs light spectrum were superior to fluorescent lamp spectrum in micro-propagation of potato plantlets. Therefore, the application of RBG was suitable; BB and RB could be used as altatives.