Abstract: Due to significant genetic differences among clones of Eucalyptus cloeziana and changes in physiological characteristics of the same clone at different culture stages, existing mature rooting media for elite varieties are difficult to adapt to other clones. To address the rooting bottleneck of the 'Chuanlin 73-86' clone, this study adopted a two-factor experimental design to systematically evaluate the regulatory effects of IBA and NAA, and screen the optimal ratio of transplantation substrates. 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 provid key support for large-scale production of Eucalyptus cloeziana.