Abstract: To investigate the relationship between key enzyme activity and 1,8-cineole content in the synthesis process of Eucalyptus globulus leaves, this study focuses on the leaves of Eucalyptus globulus at different developmental stages. It measures the relative content of 1,8-cineole and the activities of HMGR, DXS, IPPI, GPPS, and CinS, and conducts analysis of variance, correlation, and path analysis. The results showed that with the increase of leaf maturity of Eucalyptus globulus, the relative content of 1,8-cineole showed an increasing trend, while the enzyme activities of HMGR, DXS, GPPS, and IPPI showed a decreasing trend. During the complete annual growth cycle, the relative content of 1,8-cineole was significantly higher in July than in other months, and the activities of HMGR, DXS, GPPS, and CinS were significantly higher in May and June than in other months. The relative content of 1,8-cineole is significantly positively correlated with DXS and CinS activities, and significantly negatively correlated with HMGR, IPPI, and GPPS enzyme activities. The research results indicate that enhancing the enzyme activities of DXS and CinS in young leaves of Eucalyptus globulus in May and June can effectively increase the accumulation of 1,8-cineole, thereby improving the yield and quality of eucalyptus leaf oil.