The quality of material medicine resources has had a considerable impact on the development of the health industry, which has created a bottleneck for traditional Chinese medicine (TCM). Dendrobium officinale, which has been widely used for health prevention in TCM, has become a high-nutritive health food that is strongly recommended by many white-collar workers and people who pay more attention to their health. The aim of this study was to develop a method to authenticate and evaluate D. officinale from different origins via simultaneous qualitative and quantitative analyses of flavonoid glycosides. Ultra-high-performance liquid chromatography-electrospray ionization/mass spectrometry was used for the structural elucidation of the compounds. 9 characteristic peaks, including those representing 7 flavonoid C-glycosides and 2 flavonoid O-glycosides, were identified. Additionally, the contents of 5 representative flavonoid glucosides in 25 batches of D. officinale from different sources were determined. To further investigate the different sources of the 25 batch samples, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were carried out. A study on the methodology revealed that all results were reliable. This method is an efficient tool for the rapid identification of the different geographical origins of D. officinale and provides references for the quality evaluation of other natural products.