Eucalyptus cloeziana is an important, fast-growing, precious timber species in southern China, with tissue culture being the primary method for its propagation. However, the declining proliferation coefficient of adventitious shoots with multi-generation culture is a major constraint on its rapid propagation. This study aims to address this issue through the selection of suitable explants and optimizing the plant growth regulators’ formulation during the process of shoot proliferation. In this study, we cut shoots from the 21st generation of the tissue-cultured seedlings of ‘Chuanlinzhen 7523’ into apical, middle, and basal sections, and we measured their content of endogenous hormones. The proliferation coefficient (PC) and growth coefficient (GC) of explants under different concentrations of plant growth regulators were analyzed and ranked using the PCA method. The results indicated that the shoot basal sections were the best for proliferation when treated with 0.40 mg/L BAP (6-benzylaminopurine), 0.20 mg/L NAA (naphthaleneacetic acid), and 0.20 mg/L TDZ (thidiazuron), yielding a PC of 4.7 and a GC of 4.1. These basal sections ranked first with a rooting rate of up to 80%. When the rooted plantlets were acclimatized in the nursery, a survival rate of 100% was achieved. This protocol—from proliferation to acclimation—effectively improves the propagation efficiency of E. cloeziana ‘Chuanlinzhen 7523’ after multi-generation propagation.