Dendrobium nobile alkaloids (DNLA) and glycosides are the main active components extracted from Dendrobium nobile Lindl. (D. nobile) used for thousands of years in China. The pharmacological effects of the above chemical components are significantly different. D. nobile is mainly grown at an altitude ranging from 230 to 800 m in Chishui City, Northwest Guizhou Province. However, it is unclear whether the metabolite in D. nobile is influenced by the planting altitude. Hence, to reveal the different metabolite in D. nobile cultivated at the altitude of 336 m, 528 m, and 692 m, ultra-high performance liquid chromatography with Q/TOF-MS couple with multivariate analysis were developed. Using the orthogonal partial least squares-discriminant analysis, 19 different metabolites were discovered and then tentatively assigned their structures as alkaloids and glycosides by comparing mass spectrometry data with in-house database and literature. Moreover, the result of semiquantitative analysis showed the content of dendrobine that was belonged to alkaloids significantly increased at the altitude of 692 m, whereas the content of glycosides demonstrated an accumulation trend at the altitude of 528 m. The results could provide valuable information for the optimal clinical drug therapeutics and provide a reference for quality control. © 2022 The Author(s)