Although early recognition of sepsis is essential for timely treatment and can improve sepsis outcomes, no marker has demonstrated sufficient discriminatory power to diagnose sepsis. This study aimed to compare gene expression profiles between patients with sepsis and healthy volunteers to determine the accuracy of these profiles in diagnosing sepsis and to predict sepsis outcomes by combining bioinformatics data with molecular experiments and clinical information. We identified 422 differentially expressed genes (DEGs) between the sepsis and control groups, of which 93 immune-related DEGs were considered for further studies due to immune-related pathways being the most highly enriched. Key genes upregulated during sepsis, including S100A8, S100A9, and CR1, are responsible for cell cycle regulation and immune responses. Key downregulated genes, including CD79A, HLA-DQB2, PLD4, and CCR7, are responsible for immune responses. Furthermore, the key upregulated genes showed excellent to fair accuracy in diagnosing sepsis (area under the curve 0.747–0.931) and predicting in-hospital mortality (0.863–0.966) of patients with sepsis. In contrast, the key downregulated genes showed excellent accuracy in predicting mortality of patients with sepsis (0.918–0.961) but failed to effectively diagnosis sepsis. In conclusion, bioinformatics analysis identified key genes that may serve as biomarkers for diagnosing sepsis and predicting outcomes among patients with sepsis.