Abstract: To clarify the decline characteristics of tissue culture proliferative seedlings of Eucalyptus cloeziana during continuous subculture and establish an efficient rejuvenation technology system, 10 Chuanlin series clones were selected as materials in this study. The dynamic changes of multiplication coefficient, seedling morphology and abnormal traits during the subculture process were systematically monitored, and the formula of rejuvenation and rooting was optimized with the typical decling clone "Chuanlin 73-86" as the model. The results showed that although there were differences in the number of proliferation decline generations among different clones, the change law was consistent: the proliferation ability significantly decreased after 15 consecutive subcultures. For "Chuanlin 73-86", the optimal rejuvenation medium was WPM + 0.2 mg/L 6-BA + 0.2 mg/L NAA + 0.2 mg/L GA3 + 30 g/L sucrose, with an average multiplication coefficient of 3.17. After rooting, the rejuvenated seedlings had roots with dense root hairs and strong toughness; the seedlings grew vigorously, and the seedling quality met the standard. The decline law revealed and the rejuvenation technology established in this study provide key technical support for the large-scale and stable production of Eucalyptus cloeziana tissue culture seedlings.