Hashimoto's thyroiditis (HT) is a type of autoimmune disorder with a complex interplay between immune disorder and oxidative stress (OS). This research aimed to discover biomarkers and potential treatment targets associated with immune and OS dysregulation in HT through integrated bioinformatics analysis and clinical validations. Differential gene expression analysis of GSE138198 dataset from the GEO database identified 1490 differentially expressed genes (DEGs) in HT, including 883 upregulated and 607 downregulated genes. Weighted gene co-expression network analysis explored module genes associated with HT. Overlapping the differentially expressed module genes with immune-related and OS-related genes identified eight differentially expressed module genes associated with immune and OS (DEIOGs) in HT. Protein-protein interaction network analysis identified five hub genes (TNFAIP3, FOS, PTK2B, STAT1, and MMP9). We confirmed four hub genes (TNFAIP3, PTK2B, STAT1 and MMP9) in GSE29315 dataset and clinical thyroid samples, which showed high diagnostic accuracy (AUC >0.7) for HT. The expression of these four genes was positively correlated with serum thyroid peroxidase antibody, thyroglobulin antibody levels, and inflammatory infiltration scores in clinical thyroid samples. Immune profiling revealed distinct profiles in HT, such as B cells memory, monocytes and macrophages. Additionally, all hub genes were inversely associated with monocytes. Further, miRNA-mRNA network analysis was conducted, and a regulatory network comprising four hub genes, 238 miRNAs and 32 TFs was established. These findings suggest that immune cells play a crucial role in the development of HT, and the hub genes TNFAIP3, PTK2B, STAT1, and MMP9 may be key players in HT through immune- and OS-related signaling pathways. Our results may provide valuable insights into the pathogenesis and therapeutic monitoring of HT.Copyright © 2023. Published by Elsevier Inc.