This study investigates the biomarkers associated with oxidative stress and ferroptosis in ischemic stroke (IS). Differential expression analysis identified 8287 differentially expressed mRNAs (DEmRNAs) between IS and control groups. Weighted gene co-expression network analysis (WGCNA) was used to identify critical module genes, and 30 intersecting mRNAs were screened. Machine learning algorithms (LASSO and SVM-RFE) were applied to identify four biomarkers: CDKN1A, GPX4, PRDX1, and PRDX6. These biomarkers showed high diagnostic accuracy in both training and validation datasets. Gene set enrichment analysis (GSEA) revealed that these biomarkers are associated with steroid biosynthesis and immune cell infiltration. The study also constructed a miRNA-mRNA-TF regulatory network and identified 3747 regulatory pairs. Additionally, the therapeutic sensitivity of 50 drugs was analyzed, and the expression levels of the biomarkers were verified using qRT-PCR. The findings provide new insights into the pathogenesis of IS and suggest potential diagnostic and therapeutic targets.This study investigates the biomarkers associated with oxidative stress and ferroptosis in ischemic stroke (IS). Differential expression analysis identified 8287 differentially expressed mRNAs (DEmRNAs) between IS and control groups. Weighted gene co-expression network analysis (WGCNA) was used to identify critical module genes, and 30 intersecting mRNAs were screened. Machine learning algorithms (LASSO and SVM-RFE) were applied to identify four biomarkers: CDKN1A, GPX4, PRDX1, and PRDX6. These biomarkers showed high diagnostic accuracy in both training and validation datasets. Gene set enrichment analysis (GSEA) revealed that these biomarkers are associated with steroid biosynthesis and immune cell infiltration. The study also constructed a miRNA-mRNA-TF regulatory network and identified 3747 regulatory pairs. Additionally, the therapeutic sensitivity of 50 drugs was analyzed, and the expression levels of the biomarkers were verified using qRT-PCR. The findings provide new insights into the pathogenesis of IS and suggest potential diagnostic and therapeutic targets.