Abstract: In a multilayer seedling culturing system, the racks interrupt natural sunlight irradiation, it restrains the proper growth of tobacco seedlings, therefore different additional light source combinations were arranged between layers to study their influences on the growth of seedling root systems. The results showed that: 1) The activity, total absorption area, active absorption area and matter accumulation of seedling root systems were promoted by additional light irradiation; within the light radiation intensity of 19.5-92.6 μmol·m^-2·s^-1, these physiological indexes of root system positively correlated to light radiation intensity after the emergence of the third true leaf. 2) Appropriate ratio of blue/red LED emitters was 2:1, power16 W, the distance between light source and tobacco seedlings 50 cm; under such conditions, the activity of root system was 73.5% higher at the third true leaf emergence stage comparing with the treatment without light addition(CK2), which was still 10.6% higher at the sixth true leaf emergence stage; at the stage the second to the sixth true leaves emergence, the total absorption area of root system was 20.1%-36.2% higher, and the active absorption area was 37.5%-56.2%higher than CK2; at the stage the fourth to the sixth true leaves emergence, the fresh weight of roots was 11.6%-22.7% higher, and the dry weight was 11.3%-20.1% higher than CK2.