Abstract: To investigate the effects of light quality on the chloroplast pigments,light responses and root system development of tobacco seedlings in a multilayer tray seedling system,four treatments of white light(CK),red light addition(RLA),blue light addition(BLA) and LED light(LL) were experimented in a closed room.The results showed that RLA and BLA increased the content of photosynthetic pigments and the photosynthetic capacity of tobacco seedlings.In the four treatments,BLA promoted the root system development significantly,the length,surface area,average diameter and volume of root were 75.45 cm,13.49 cm²,0.51 mm and 0.18 cm³,respectively,at the stage of 4-leaf and 1-bud of seedlings.Photosynthetic capacity of leaves and root system development significantly positively correlated to the accumulation of dry matter in tobacco seedlings in multilayer seedling system.The analysis results indicated that blue light addition was more beneficial to the cultivation of flue-cured tobacco seedlings in a multilayer tray seedling system.