Ophiocordyceps sinensis, a medicinal fungus utilized in traditional Chinese medicine, exhibits a range of biological activities and pharmacological functions. In this study, we determined the amino acid composition of 94 amino acids in Ophiocordyceps sinensis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fresh samples of Ophiocordyceps sinensis were analyzed under three different drying methods: vacuum freeze drying (DG), oven drying (HG), and air drying (YG). This investigation aims to assess the effects of these drying methods on the content and quality of amino acid metabolites in Ophiocordyceps sinensis. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed for sample classification and the identification of differentially accumulated metabolites (DAMs). The results revealed the detection of 79 amino acid metabolites, which included elevated levels of oxidized L-glutamic acid, L-glutamic acid, and glutathione. Differential amino acid metabolites that met the criteria of fold change (|FC|) ≥ 2, p-value (p) ≤ 0.5, and variable importance in projection (VIP) ≥ 1 were analyzed. Significant differences in 48 amino acid metabolites between the groups were primarily related to protein synthesis. According to the KEGG analysis, all three comparison samples exhibited significant enrichment in several pathways. These pathways included the interaction of neuroactive ligands with receptors, the metabolism of cysteine and methionine, and the biosynthesis of plant hormones. The variations in amino acid metabolite levels observed across the three drying methods may be attributed to the degradation of proteins or amino acid metabolites, influenced by several factors, including temperature, enzyme activity, and moisture content. Additionally, Maillard and oxidative reactions involving substances such as amino acids, sugars, and oxygen may also play a significant role. This study demonstrates that various drying methods significantly influence the amino acid metabolite content of Ophiocordyceps sinensis. Therefore, the selection of drying methods should be tailored to meet specific requirements. This research provides important insights into the metabolite composition of Ophiocordyceps sinensis under different drying techniques, thereby contributing to a more comprehensive understanding of its nutritional and therapeutic properties.