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The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered <i>Dendrobium catenatum</i> as a chassis plant for the production of dendrobine through the screening and pyramiding of key biosynthesis genes. Initially, previously predicted upstream key genes in the methyl-D-erythritol 4-phosphate (MEP) pathway for dendrobine synthesis, including <i>4-(Cytidine 5′-Diphospho)-2-C-Methyl-d-Erythritol Kinase</i> (<i>CMK</i>), <i>1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase</i> (<i>DXR</i>), <i>2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase</i> (<i>MCT</i>), and <i>Strictosidine Synthase 1</i> (<i>STR1</i>), and a few downstream post-modification genes, including <i>Cytochrome P450 94C1</i> (<i>CYP94C1</i>), <i>Branched-Chain-Amino-Acid Aminotransferase 2</i> (<i>BCAT2</i>), and <i>Methyltransferase-like Protein 23</i> (<i>METTL23</i>), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes (<i>SG</i>) were then stacked and transiently expressed in the leaves of <i>D. catenatum</i>, resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control (<i>EV</i>). Further, RNA-seq analysis identified <i>Copper Methylamine Oxidase</i> (<i>CMEAO</i>) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of <i>CMEAO</i> increased dendrobine content by two-fold. Additionally, co-expression analysis of the differentially expressed genes (DEGs) by weighted gene co-expression network analysis (WGCNA) retrieved one regulatory transcription factor gene <i>MYB61</i>. Overexpression of <i>MYB61</i> increased dendrobine levels by more than two-fold in <i>D. catenatum</i>. In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of <i>D. catenatum</i> to produce dendrobine, thereby improving its medicinal value.