Pentacyclic triterpenes are isoprene-derived secondary metabolites that are synthesized by the cyclization of 2,3-oxidosqualene. Two pentacyclic triterpenoids, taraxasterol and ψ-taraxasterol, are particularly rich in dandelion and are useful candidates for pharmacologically active triterpenes. A multifunctional oxidosqualene cyclase (TcOSC1) of Taraxacum coreanum catalyzes the cyclization of 2,3-oxidosqualene into various triterpenes (taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, α-amyrin, and dammarenediol-II) which were demonstrated by heterologous expression in the yeast. Here, we evaluated the production of taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, and α-amyrin in transgenic tobacco overexpressing the T. coreanum TcOSC1 gene. Transgenic tobacco plants were obtained via Agrobacterium tumefaciens-mediated transformation. The introduction and expression of transgenic genes in tobacco was confirmed by genomic PCR and qRT–PCR, respectively. All lines of transgenic tobacco produced five new triterpenes, namely, taraxasterol, ψ-taraxasterol, δ-amyrin, β-amyrin, and α-amyrin, but not dammarenediol-II. The accumulation of phytosterols (β-sitosterol, campesterol, and stigmasterol) was reduced in transgenic tobacco compared to the wild-type control. Triterpene accumulation in transgenic tobacco plants was different among leaf, stem, and root. The highest level of triterpenes (617 µg g⁻¹ dry weight) were observed in leaf of Tr4 transgenic line. Conclusively, we established the production of multiple triterpenes in transgenic tobacco by overexpressing the TcOSC1 gene, which can be applied in the production of triterpenes for utilization as a source of pharmacologically active materials.