Abstract: In order to explore the optimal conditions for the rooting of tissue culture seedlings of Acacia melanoxylon, on tissue culture seedlings of superior clones A. melanoxylon, the study was to explore the effects of the rooting promoter, MS mother liquor and its dosage, dark treatment time, temperature and organic additives on its rooting condition in the single-factor experimental design and thus propose a rooting technique system of tissue culture seedlings on A. melanoxylon clones, indicating that IBA and ABT-1 were the rooting promoter with the best treatment; 1/2MS improvement (2) was registered the best mother liquor for rooting; the dark treatment should be performed for 4 days; 25 ℃ offered the best tissue culture; rooting rate was 96.63%; and the rooting of organic additives such as tryptone, banana and coconut milk was inferior to the control.