Abstract: In order to analyze the difference in metabolite composition between wolfberry leaves black tea, which is produced from wolfberry leaves by using black tea fermentation techniques, and the traditional black tea, the metabolites in both types of tea were quantitatively determined and identified by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with fully targeted metabolomics technology. The results showed that a total of 447 and 381 metabolites were screened from wolfberry leaves black tea and traditional black tea, respectively. Among them, there were 223 common metabolites, and 224 and 158 unique metabolites, respectively, indicating a significant distinction in their metabolic profiles. The multivariate statistical analysis further confirmed a clear separation in the overall composition of metabolites between the two teas. In terms of the relative content of metabolite categories, the phenylpropanoids and phenolic compounds in wolfberry leaves black tea were relatively enriched, while the traditional black tea showed more abundant terpenoids and flavonoid components. At the level of specific markers, the relative contents of L-lysine, tryptophan, 3,4-dihydrocoumarin, esculetin, isoeugenol, geranyl acetate, β-ionone, and safranal in wolfberry leaves black tea were significantly higher than those in the traditional black tea. In contrast, the contents of histidine, diadinoxanthin, coumarin, and quinic acid were higher in the traditional black tea. The study revealed the systematic differences in the material basis between wolfberry leaves black tea and traditional black tea from the metabolomics perspective, and clarified their characteristic compositional profiles, which provided a chemical basis for the quality evaluation of wolfberry leaves black tea as a novel functional tea beverage.