Abstract: Due to significant genetic differences among different clones of Eucalyptus cloeziana, as well as changes in physiological characteristics of the same clone at different culture stages, the existing mature rooting media designed for elite varieties are not suitable for other clones. To address the rooting bottleneck of 'Chuanlin 73-86' clone, a two-factor experimental design was employed to systematically evaluate the regulatory effects of IBA and NAA, with the aim of determing the optimal ratio for transplantation medium. The results showed that IBA, NAA, and their interaction all significantly affected the rooting effect: the rooting rate exceeded 60% when IBA was added alone, while the combined addition of IBA and NAA further optimized the rooting rate. Among the tested formulas, 1/2MS + 1.00 mg/L IBA + 0.25 mg/L NAA was the optimal one, achieving a rooting rate of 91.63% with robust roots and well-developed root hairs. Transplantation experiments indicated that the substrate ratio of peat soil: perlite: coconut coir = 2:1:1 yielded the highest survival rate (91.44%), and the seedlings grew vigorously. These results provide key support for large-scale production of E. cloeziana.