Objective This study aimed to investigate the potential value of serum hypoxia-inducible factor-1α (HIF-1α) concentrations as a biomarker in patients with sepsis. Methods The enrolled patients were divided into the following four groups: the intensive care unit (ICU) control group (n = 33), infection group (n = 29), septic nonshock group (n = 40), and septic shock group (n = 94). An enzyme-linked immunosorbent assay was used to measure serum HIF-1α concentrations on ICU admission. Clinical parameters and laboratory test results were also collected. Results Serum HIF-1α concentrations were significantly higher in the infection group, septic nonshock group, and septic shock group than in the ICU control group. Moreover, HIF-1α concentrations were associated with a better predictive ability for diagnosing sepsis than the Acute Physiology and Chronic Health Evaluation II score, procalcitonin concentrations, and lactate concentrations. Patients with sepsis and HIF-1α concentrations >161.14 pg/mL had a poor prognosis. Conclusions Serum HIF-1α concentrations are a useful biomarker for the diagnosis of sepsis and predicting the prognosis of patients.