Abstract: The fermentation of lactic acid bacteria can enhance the transformation of active substances, such as polyphenols in food. This study aims to explore the effects of lactic acid bacteria fermentation on their functional activities and phenolic compounds in Dendrobium officinale (DO). Three commonly used lactic acid bacteria were first selected to ferment the DO from five regions. Then, a systematic investigation was implemented to determine the total phenolic and flavonoid contents and the antioxidant activity of DO before and after fermentation. The phenolic components and metabolite composition were finally characterized using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the viable cell counts gradually increased in the DO fermentation broth, as the fermentation progressed, whereas, there was a gradual decrease in the pH value. The growth rate of viable cells was highest during the first 0 to 24 h fermentation, as the decline rate of pH value. All three lactic acid bacteria enhanced the contents of total phenolic and total flavonoid, as well as the antioxidant activity of DO. The most significant enhancement was observed to ferment with Lactobacillus bulgaricus for 48 h in the DO in Wenshan City, Yunnan Province. The total phenolic and total flavonoid contents of the fermented DO were 120.67 and 23.39 mg/g, respectively, under the same condition. Additionally, the radical scavenging abilities against the cationic ABTS radical (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (1,1-diphenyl-2-picrylhydrazyl) as well as the ferric reducing ability (FRAP) were highest at 288.51, 34.34, and 136.32 mg/g, respectively, which increased of 0.81, 2.11, and 0.57 times than before fermentation. 16 phenolic compounds were selected before and after DO fermentation using HPLC. Specifically, the content of sinapic acid increased from 0.65 to 1.14 mg/g (an increase of 75.38%), while the content of vanillic acid rose from 2.60 to 3.19 mg/g (an increase of 22.70%). However, the content of (-)-epicatechin gallate decreased from 2.92 to 0.47 mg/g (a decrease of 83.90%). Moreover, the salicylic acid was only detected in the post-fermentation samples. Non-targeted metabolomics analysis revealed that a total of 1,298 metabolites were identified from Dendrobium officinale before and after fermentation. Among them, 100 and 85 metabolites were significantly upregulated in the positive and negative ion modes, respectively, while 211 and 180 metabolites were significantly downregulated. The number of upregulated metabolites was notably lower than that of downregulated metabolites. The upregulated metabolites were primarily concentrated in the fatty acids, nucleosides, and alkaloids, whereas, the downregulated metabolites were found mainly in the flavonoids, phenolic compounds, and terpenes. It infers that these metabolites were gradually consumed or metabolically transformed during fermentation. Correlation analysis demonstrated a high correlation between antioxidant activity and phenolic compounds. Enrichment analysis of metabolic pathways was performed during Dendrobium officinale fermentation using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The most likely metabolic pathways were the ABC transport pathway, biosynthesis of various secondary metabolites - part II, and metabolism of alanine, aspartate, and glutamate. In conclusion, the fermentation of lactic acid bacteria significantly improved the antioxidant activity of DO, especially Lactobacillus bulgaricus. This improvement was closely related to the transformation and release of phenolic compounds during fermentation. Dendrobium officinale can be expected to serve as the biotransformation of active components in natural products/foods.