Cancer remains a leading cause of mortality worldwide, with human exonuclease 1 (EXO1) emerging as a key player in DNA repair and damage response pathways, critical for genomic stability and tumor evolution. The aim of this study was to conduct a comprehensive pan-cancer analysis to elucidate the multifaceted roles of EXO1 in various malignancies. Leveraging public databases including TCGA, GTEx, HPA, cBioPortal, UALCAN, STRING, CancerSEA and TISIDB database, we examined EXO1's expression, diagnostic potential, prognostic significance, mutational characteristics, functional roles, and immunological effects across different cancer types. EXO1 was found to be upregulated in multiple cancers, with significant diagnostic potential as indicated by high AUC values in ROC analyses. Elevated EXO1 expression correlated with adverse prognosis in several cancer types, including breast, lung, and pancreatic cancers. Epigenetic alterations, including DNA methylation and mRNA modifications, were also associated with EXO1 expression. Enrichment analyses identified EXO1-related genes involved in DNA recombination, replication, and repair, with GSEA implicating EXO1 in cell cycle regulation and DNA processing pathways. Importantly, immunogenomic analyses revealed EXO1's significant role in modulating the tumor microenvironment, as it is associated with immune cell infiltration and cytokine expression, suggesting its involvement in tumor immunology and immune response regulation. These results implied that EXO1 as a significant biomarker with prognostic and diagnostic potential across various malignancies, suggesting its potential as a therapeutic target and its involvement in immunomodulatory processes within the tumor microenvironment.