Sclareol, a diterpene alcohol, is the most common starting material for the synthesis of ambrox, which serves as a sustainable substitute for ambergris, a valuable fragrance secreted by sperm whales. Sclareol has also been proposed to possess antibacterial, antifungal, and anticancer activities. However, in nature, sclareol is only produced by a few plant species, including Cistus creticus, Cleome spinosa, Nicotiana glutinosa, and Salvia sclarea, which limits its commercial application. In this study, we cloned the two genes responsible for sclareol biosynthesis in S. sclarea, labda-13-en-8-ol diphosphate synthase (LPPS) and sclareol synthase (SS), and overexpressed them in tobacco (Nicotiana tabacum L.). The best transgenic tobacco lines accumulated 4.1 μg/cm2 of sclareol, which is comparable to the sclareol production of N. glutinosa, a natural sclareol producer. Thus, sclareol synthesis in tobacco represents a potential alternative means for the production of this high-value compound.