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Gastrodin (GAS), the main bioactive component of G. elata Blume, has important pharmaceutical and functional activities. The aim of this study is to produce GAS from p-2-hydroxybenzyl alcohol (HBA) through biotransformation. The conversion of exogenous HBA into GAS compound was conducted using cell suspension cultures of Armillaria luteo-virens Sacc. The bioconversion conditions were fully optimized with response surface methodology (RSM), turning out that the optimal transformation conditions composed of 3 mg/mL HBA, 6.5 g/30 mL inoculums level, 1.5% Tween 80, pH 4.5, and transformation temperature at 23°C. Under the optimized conditions, the conversion productivity of GAS reached the highest value (5.65 ± 0.45 mg/L). Verified experiments further validated that the optimized conditions were suitable for predicting the actual process of HBA transformation in the resting-cell system. The bioconversion kinetics model was as well simulated with Michaelis–Menten equation, which showing the suitability. The present study proposed the biotransformation pathway of HBA into GAS by resting cells transformation, indicating that the biotransformation process involved glucosylation reaction. Furthermore, Imprinting Control Region (ICR) mice in vivo demonstrated that the identified gastrodin possessed a significant anti-inflammatory activity. The fundamental data in the present work provides an efficient way to produce GAS through the whole-cells biocatalysis.