Dalbergia odorifera T. Chen, endemic to China, is the sole plant source for the valuable Chinese medicine Dalbergiae Odoriferae Lignum. Its heartwood, naturally forming over 40 years, is rare due to low formation rates and a scarcity of mature trees. Artificial induction of heartwood formation using ethylene shows promise, though its mechanisms remain unclear. This study investigates the regulation of heartwood biosynthesis by screening related signal factors, constructing and analyzing the transcriptome, and validating findings through suspension systems. Key results include: Ethylene is an effective endogenous hormone involved in regulating heartwood-like substances in D. odorifera. Ethephon (ETH) treatment enhanced heartwood characteristics, increasing ethylene synthesis and secondary metabolite production. After 14 days of ETH treatment, branches maintained healthy growth, and heartwood color matched natural heartwood. ETH and H2O2 promoted endogenous ethylene synthesis, enhancing overall metabolism, tree resistance, and enzyme activities. Inhibiting ethylene synthesis prevented heartwood color change and secondary metabolite accumulation. The ethylene signaling pathway is crucial for heartwood substance synthesis. Transcriptome analysis revealed that differentially expressed genes (DEGs) were enriched in pathways related to cysteine and methionine metabolism, phytohormone signaling, and secondary metabolite synthesis. Significant DEGs in the ethylene signaling pathway included upregulated ACO, EIN2, EIN3, and ERF1/2 in transition regions. qRT-PCR validation showed upregulated secondary metabolite synthesis genes (DoNES1, DoCHS1) and consistent expression patterns of ethylene signaling genes (DoACO1, DoEIN3-1). This research enhances understanding of heartwood formation mechanisms in D. odorifera and supports the development of artificial heartwood cultivation techniques.