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 Dendrobium catenatum 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 4-(Cytidine 5′-Diphospho)-2-C-Methyl-d-Erythritol Kinase (CMK), 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (DXR), 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (MCT), and Strictosidine Synthase 1 (STR1), and a few downstream post-modification genes, including Cytochrome P450 94C1 (CYP94C1), Branched-Chain-Amino-Acid Aminotransferase 2 (BCAT2), and Methyltransferase-like Protein 23 (METTL23), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes (SG) were then stacked and transiently expressed in the leaves of D. catenatum, resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control (EV). Further, RNA-seq analysis identified Copper Methylamine Oxidase (CMEAO) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of CMEAO 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 MYB61. Overexpression of MYB61 increased dendrobine levels by more than two-fold in D. catenatum. In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of D. catenatum to produce dendrobine, thereby improving its medicinal value. © 2023 by the authors.