Background Sepsis is a serious syndrome that is caused by immune responses dysfunction and leads to high mortality. The abilities of heat shock protein 90α (HSP90α) in assessing the diagnosis and prognosis in patients with sepsis remain ill-defined to date. We conducted a study to reveal the possible clinical applications of HSP90α as biomarker for the diagnosis and prognosis in patients with sepsis. Methods In total, 150 patients of sepsis, 110 patients without sepsis admitted to ICU and 110 healthy subjects were involved in this study. The serum HSP90α contents, sequential organ failure assessment (SOFA) scores, procalcitonin (PCT), and short-term survival status of the participants were measured and compared. Logistic and linear regression models adjusting for potential confounders were used to examine the association of HSP90α with sepsis survival. Moreover, serum IL-1β, IL-18, MIP-3α, and ENA-78 were also determined. Finally, Spearman correlation analysis was employed to reveal a possible mechanism that HSP90α contributed to the short-term deaths. Results Serum HSP90α levels in sepsis patients were higher than those in ICU controls and healthy controls (P < 0.001), and even increased in patients who died within 28 days (P < 0.001). Logistic and linear regression models identified HSP90α was an independent risk factors for sepsis mortality. Receiver operating characteristic (ROC) analysis displayed that HSP90α had a considerable predictive performance for sepsis outcome, with an area under curve (AUC) value up to 0.79. Survival analysis demonstrated that the mortality of sepsis individuals at 28 days was positively associated with HSP90α levels, especially the levels of HSP90α were greater than 120 ng/mL (P < 0.001). Moreover, among sepsis patients, those who died had notably elevated cytokines, IL-1β, IL-18, and chemokines, MIP-3α, ENA-78, relative to survivors. Further correlation analysis demonstrated that there was a nominally positive correlation between HSP90α and IL-1β, IL-18, and MIP-3α. Conclusion HSP90α is of favorable clinical significance in sepsis diagnosis and prognosis, laying a foundation for future clinical applications.