Abstract: This study aimed to promote the release of active compounds in Dendrobium officinale and enhance its antioxidant activity. Lactiplantibacillus plantarum (L. plantarum), Bacillus amyloliquefaciens (B. amyloliquefaciens), and Saccharomyces cerevisiae (S. cerevisiae) were used for single-strain fermentation of D. officinale, and the optimal fermentation strains were selected based on total antioxidant capacity. The results showed that L. plantarum G35, B. amyloliquefaciens MMB-02, and S. cerevisiae A8 exhibited the highest antioxidant capacities at fermentation times of 24, 48, and 72 hours, with values of 4.13±0.08, 3.94±0.08, and 3.88±0.04 Fe²⁺ mmol/L, respectively. Additionally, the polyphenol and total flavonoid contents in the fermentation supernatant of D. officinale increased significantly (P<0.05), with the L. plantarum G35 fermentation group showing the highest levels of polyphenols and flavonoids at 50.89±1.28 μg/mL and 110.29±2.79 μg/mL, respectively, after 24 hours of fermentation. Further optimization of the mixed-strain fermentation process by applying the response surface methodology, the optimal fermentation conditions for D. officinale were determined as follows: Fermentation time 43.0 hours, temperature 32.0 ℃, shaking speed 180 r/min, and initial pH7.0. Under these conditions, the total antioxidant capacity of the fermented D. officinale reached 5.04±0.03 Fe²⁺ mmol/L. These findings provide theoretical support for improving the antioxidant activity of D. officinale and offer a reference for enhancing its bioavailability.