〔Abstract〕 Objective The thin-layer identification of main medicinal tastes of Tianma Anshen Decoction and the simultaneous determination of gastrodin and p-hydroxybenzyl alcohol in Tianma Anshen Decoction were established for the accurate quality control and evaluation of the prescription. Methods The identification of Gastrodia and Nightfork was performed by thin layer chromatography (TLC), and the content was determined by high performance liquid chromatography (HPLC). Agilent TC C18 column was used as stationary phase, and acetonitrile-0.05% phosphoric acid solution (3:97) as the mobile phase, the flow rate was 1.0 mL·min^-1, the detection wavelength was 220 nm, the column temperature was 25 ℃, and the sample size was 10 μL. 10 batches of Tianma Anshen decoction were prepared by parallel preparation of gastrodin and p-hydroxybenzyl alcohol. Results The results of 10 batches of samples showed that the content of gastrodin was 0.0311 ~ 0.0365 mg·mL^-1 and p-hydroxybenzyl alcohol was 0.0210 ~ 0.0245 mg·mL^-1. Conclusion The simultaneous determination of gastrodin and p-hydroxybenzyl alcohol in Gastrodia is carried out. The method is rapid, accurate and efficient, which provided scientific basis for the precise quality control of the prescription.