This study investigated the effects of 2,4-epibrassinolide (EBR) residues on Dendrobium officinale quality. The residues decreased from 0.164 to 0.238 mg/kg (2 h) to 0.014–0.071 mg/kg (35 d) in the stems with 0.02–0.05 mg/L EBR applications. Polysaccharide, mannose, and glucose content were increased in the stems and leaves, reaching a maximum of 61% in dried stems on 35 d. The increase in content may result from the significant upregulation of enzyme activities, including UGP, SUS, and SPS. Further analysis by nontargeted metabolomics revealed 5 upregulated (UDP-l-rhamnose, mannose-6-phosphate, GDP-mannose, chitobiose, and N-acetyl-galactosamine-6-phosphate) and 13 downregulated metabolites associated with polysaccharide and monosaccharide growth in the 0.02–0.05 mg/L EBR treatments. These differential metabolites regulate the biosynthesis of polysaccharides mainly through key metabolic pathways, such as glucose and fructose metabolism. These results provide a reference for the regulatory mechanisms governing the quality indicators of Dendrobium officinale after EBR application.