Abstract Dalbergia odorifera T. Chen, an economically and medicinally valuable tree, suffers from heartwood shortage due to slow natural formation. Ethylene has emerged as a potent inducer of heartwood formation in D. odorifera, but long-term evidence and mechanisms remain unclear. To bridge this gap, we systematically assessed dynamic changes in morphology, anatomy, metabolomics and antimicrobial properties of ethylene-induced heartwood zone over 1, 3 and 5 years, comparing it with 20-year-old natural heartwood. Results showed that in the ethylene-stimulated zone, heartwood resins were initially deposited at the outermost layer and progressively infiltrated inward until full saturation. The coloration of induced heartwood zone gradually intensified, eventually developing hues and grain patterns resembling natural heartwood by 5 years. Heartwood extracts yield and antimicrobial activity matched natural heartwood after 1 year. Total flavonoid content matched natural heartwood by 3 years, with nine flavonoid components (e.g. naringenin, formononetin) governing color development. While volatile compounds composition matched natural heartwood after 5 years, flavonoid profiles remained distinct, indicating volatile compounds biosynthesis preceded flavonoids. More interestingly, this study proposed a novel hypothesis for heartwood formation: during natural heartwood development, a certain factor (e.g. ethylene) induces a transition zone containing numerous ‘micro-zones,’ where cell death and heartwood substance deposition progress gradually from the outer to inner layers. This pattern leaves residual sapwood within developing heartwood, effectively resolving the theoretical conflict regarding the presence of living cells in heartwood. Thus, this study not only confirms the long-term feasibility of ethylene-induced heartwood zone formation and its underlying mechanisms, but also proposes a groundbreaking hypothesis that challenges conventional understanding of heartwood formation, laying a crucial foundation for future research on heartwood formation mechanisms and targeted cultivation of high-value tree species.