<p><i>Dendrobium officinale</i> is an important medicinal orchid, but the development of improved germplasm is limited by the availability of stable breeding materials. In this study, nodal stem segments from a single diploid clonal line were used to generate autotetraploid plants, establish a one-step plantlet regeneration system, and examine ploidy-associated transcriptomic changes. Stem segments were treated with 0.05% colchicine under a time-gradient regime, and candidate plants showing thickened leaves, darker leaf color, and shortened internodes were further screened by flow cytometry and confirmed by chromosome counting. A stable autotetraploid line was obtained under the 228 h treatment, with a chromosome number of 2n = 4x = 76 maintained over successive subcultures. An optimized one-step regeneration medium enabled simultaneous shoot proliferation and rooting from tetraploid stem segments, with a multiplication coefficient of 10.11 times. In addition to axillary bud regeneration, some tetraploid explants regenerated through protocorm-like bodies, providing an additional propagation route. Compared with the diploid source line, the autotetraploid line showed greater shoot height and leaf area, larger stomata, and lower stomatal density, whereas overall tissue organization remained largely unchanged. Transcriptome analysis identified 3740 differentially expressed genes between diploid and autotetraploid leaves, with significant enrichment in plant hormone signal transduction and metabolic pathways. These results establish an integrated route for autotetraploid induction, verification, rapid propagation, and comparative analysis in <i>D. officinale</i>, and provide a basis for the creation and further evaluation of autotetraploid germplasm.</p>