Abstract: To clarify the decline characteristics of tissue culture proliferative seedlings of Eucalyptus cloeziana during continuous subculture and establish an efficient rejuvenation technology system, this study used 10 Chuanlin series clones as materials. It systematically monitored the dynamic changes of multiplication coefficient, seedling morphology and abnormal traits during the subculture process, and took the clone "Chuanlin 73-86" (with typical decline characteristics) as a model to optimize the rejuvenation and rooting formulas.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.