Objective Dendrobium officinale has long been used as an important medicinal herb in oriental medicine. Polysaccharide, flavonoid, and alkaloid are the major active ingredients, the production and accumulation of which are frequently affected by numerous environmental cues. Phytohormone supplemented in culture medium has facilitated the mass production of orchids. However, their mechanism of action on the production of active components in Dendrobium officinale is far from clear.Methods Here, major medicinal metabolites were comparatively analyzed in Dendrobium officinale seedlings exposed to the most commonly used phytohormones (NAA and/or 6-BA), and transcriptomes corresponding to the treatments were generated.Results Results showed that phytohormones greatly affected the accumulation of polysaccharide, alkaloid, and flavonoid, and triggered tremendous transcriptional changes. It was demonstrated that 6-BA induced more transcripts than NAA and that β-glucosidase (BGLU) expression was closely related to polysaccharide production. Further functional analysis revealed that factors including phytohormone category, concentration, treating duration, and seedling growth stages, can drastically affect the BGLU expression and the corresponding polysaccharide production, thus partially answered the key question at molecular level why the medicinal constituents are unstable in tissue culture derived Dendrobium plants.Conclusion Altogether, the present study clearly demonstrated that BGLU is a key regulator for polysaccharide production in Dendrobium officinale in response to phytohormone treatments.