Abstract The intercropping approach of Eucalyptus and native trees has been widely recommended, as an ideal replacement for monoculture Eucalyptus plantations (EUs), to ameliorate global biodiversity loss and mitigate environmental change. However, both suitable native tree species (NS) and the best intercropping ratio between Eucalyptus and native trees have not been determined. To fill this gap, a four‐level intercropping gradient of Eucalyptus urophylla planted with eight NS was set up (i.e., 20%NS, 30%NS, 40%NS, 50%NS), monitored and compared with a monoculture E. urophylla plantation (EU) and a randomly mixed plantation of nine NS in southern China. The results showed that, the intercropping ratio of Eucalyptus and native trees had a long‐term effect on tree layer structure and health status, and a cascading effect on the thermodynamic health state of soil microbes. Shade‐tolerant woody species are more suitable for intercropping with Eucalyptus. Intercropping plantations with not less than 30% native trees were more favourable for long‐term survival and growth of both planted Eucalyptus and native trees, and provided much more favourable conditions for the natural immigration of other native trees, which leads to a healthy plant community with significantly higher eco‐exergy than EU. The initial mixing ratio between Eucalyptus and high diversity native trees affected soil fertility through its long‐term effects on the biodiversity and bio‐thermodynamic state of trees and soil microbes. Synthesis and applications. Our results highlight the long‐term positive effect of the intercropping ratio of Eucalyptus and high diversity native trees on multi‐trophic biodiversity conservation, bio‐thermodynamic health development and soil fertility conservation. In the conversion of monoculture EUs to multi‐species plantations, it is recommended to mix more than 30% NS, which have different ecological niches with Eucalyptus.