The invention discloses a Herba Dendrobii tissue cultured seedling medium and a preparation method thereof. The Herba Dendrobii tissue cultured seedling medium comprises the following raw materials: watermelon juice, malic acid, o-benzoic sulfimide, tea polyphenols, sylvite, microcosmic salt, nitrogen salt, calcium salt, magnesium salt, iodine salt, boron salt, molybdenum salt, zinc salt, manganese salt, copper salt, molysite, vitamin, amino acid, indolebutyric acid, gibberellin, 6-benzyl adenine, naphthylacetic acid, kinetin, asparagine, a conditioner, a cosolvent, a dispersant, a compatilizer, a stabilizer, an antibacterial agent and water. Seedlings obtained by tissue culturing of Herba Dendrobii by the Herba Dendrobii tissue cultured seedling medium are good in quality and high in survival rate, and contents of alkaloid, polysaccharide and amino acid can be increased effectively.