Abstract: This study aimed to explore the quality differences of Dendrobium officinale from various origins and cultivation methods. A total of eight samples of Dendrobium officinale were collected from six locations: Dehong in Yunnan, Lingshan in Guangxi, Sansui in Guizhou, Huoshan in Anhui, Yueqing in Zhejiang, and Yuyao in Zhejiang. The study systematically compared the differences in basic components, active substance content, and antioxidant activity. The results indicated that the quality of Dendrobium officinale was significantly influenced by its origin and cultivation modes. The fat content of the eight samples ranged from 0.15% to 1.23%, protein content from 2.79% to 4.29%, and ash content from 2.31% to 6.23%. The total polysaccharide content of Dendrobium officinale grown in greenhouses (27.63% to 41.06%) was higher than that of those cultivated in simulated wild (26.80% to 26.96%). However, the simulated wild cultivated Dendrobium officinale exhibited superior performance in total flavonoids (0.468% to 0.513%), total phenolic content (0.347% to 0.356%), and DPPH radical scavenging ability (3.175 to 4.667 mg Trolox/g). All eight samples contained protocatechuic acid, vanillic acid, ferulic acid, and benzoic acid, with benzoic acid being the main phenolic acid component. The relative content of benzoic acid in the Guizhou Sansui (SH3) sample was the highest, reaching 14.908, which is 5.95 times that of the Zhejiang Yuyao (SH7). The total amino acid content ranged from 2413~3596 mg/100 g, and the ratio of essential amino acids to total amino acids was between 35.82% and 37.45%. Principal component analysis further confirmed that the origin and cultivation modes significantly affect the nutritional components of Dendrobium officinale, thereby influencing its quality and pharmacological effects. In conclusion, Dendrobium officinale produced in Zhejiang, particularly under wild- simulated cultivation, exhibits higher levels of total phenolics and total flavonoids along with stronger antioxidant activity, while the greenhouse cultivation mode is more conducive to the accumulation of polysaccharides.