Changes in non-volatile substances in traditional Yi ethnic fermented medicinal liquor, made from wild Rosa roxburghii, Phyllanthus emblica, lemon, Gastrodia ginseng, and black honey as the main raw materials, were examined. Solid-state natural fermentation technique was employed under constant low temperature (10±0.5 ℃) in dark. Non-targeted metabolomics was used for detailed differential analysis of non-volatile substances in traditional Yi ethnic fermented medicinal liquor before and after fermentation. A total of 208 differential metabolites from 17 categories were found in the medicinal liquor before and after fermentation (variable importance in projection >1 and P<0.05), which included 69 phenols (47 flavonoids), 31 alkaloids, 18 terpenes, and 13 nucleotides and their derivatives. Among them, phenols accounted for the largest proportion at 33.18% (22.60% flavonoids), followed by alkaloids (14.90%), terpenes (8.65%), and nucleotides and their derivatives (6.25%). Further analysis based on Kyoto Encyclopedia of Genes and Genomes metabolic pathways revealed 19 significantly different metabolic pathways involving 12 key differential metabolites. The contents of nucleotides and their derivatives, amino acids and their derivatives, and phenolic substances increased substantially during the fermentation process, thereby notably enhancing the sensory quality and nutritional value of the medicinal wine. The data support and theoretical basis provided by this study can be utilized to further explore the material changes and metabolic mechanisms in Yi traditional fermented wine during the fermentation process.