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Bacterial flora is present in various parts of the human body, and bacterial 16S rRNA genes have been detected in the bloodstream. Distinct blood microbiomes have been identified in various diseases, including cancer, and are thought to play a role in disease pathogenesis. In this study, we conducted a 16S rRNA metagenomic analysis of serum extracellular vesicles from 89 patients with gastric cancer (GC) and 25 healthy donors. We identified lower levels of Bacteroidetes and Actinobacteria and higher levels of Firmicutes in patients with GC than in healthy donors. By integrating this characteristic bacterial DNA profile, we developed a BAF index, defined as the ratio of Bacteroidetes and Actinobacteria to Firmicutes, which exhibited high sensitivity for detecting GC in both the discovery and validation cohorts, suggesting its potential utility as a screening tool. A high BAF index was significantly associated with an advanced tumor stage and poor prognosis. Moreover, a high BAF index was linked to an immunosuppressive tumor microenvironment, which may contribute to the unfavorable outcomes observed in these patients. These findings indicate that circulating bacterial signatures may serve as promising biomarkers for GC.