Abstract: Objective: To conduct an analysis of the flavonoids in Dendrobium officinale Kimura et Migo from Guangxi and to investigate the effects of different growth years on the accumulation of flavonoids. Methods: A Kromasil 100-5 C18 column (250 mm×4.6 mm, 5 μm) was used for chromatographic separation with a gradient elution of acetonitrile-methanol (v:v=1:1)−0.01 mol/L ammonium acetate solution as the mobile phase. Mass spectrometry data was collected using ESI ion source in negative ion mode. HPLC was used to establish the characteristic chromatographic fingerprints of 25 batches of Dendrobium officinale from Guangxi, and principal component analysis and cluster analysis were employed to investigate the effects of different growth periods on the accumulation of flavonoid components. Results: A total of 19 flavonoids were identified from Dendrobium officinale from Guangxi through the HPLC-ESI-MSⁿ technique, encompassing 9 flavonoid glycosides, 4 flavonol glycosides, 2 acylated flavonoid glycosides, 3 acylated flavonol glycosides, and 1 dihydroflavone. The characteristic maps of flavonoids in Dendrobium officinale from Guangxi were established, and 9 common peaks were identified. Based on clustering and principal component analysis, 25 samples were categorized into four classes in accordance with growth years and rod color (green rod for 1~3 years, green rod for 3~5 years, red rod for 1~3 years, red rod for 3~5 years). Additionally, the peak area of the characteristic peak of flavonoids increased with the increase of growth years. There was no marked difference in flavonoid content between the 'red' and 'green' during the 1~3 years of growth. Nevertheless, the contents of moscatin I, moscatin III, chrysin, and specific peaks (peak 8, peak 9) in the red rod samples were significantly higher than those in the green rod samples during the 3~5 years. Conclusion: In this study, leveraging mass spectrometry analysis integrated with chemical pattern recognition, elucidated the common characteristics of flavonoid compounds in Dendrobium officinale sourced from Guangxi. It emphasized the crucial influence of growth years on the accumulation of flavonoid compounds. Specifically, after 3~5 years of growth, the flavonoid content in 'red stem' samples was significantly higher than that in 'green stem' samples. This finding provided a solid theoretical basis for the investigation of pharmacological activities, application development, evaluation of genetic resources, and optimization of harvesting strategies for Dendrobium officinale.