Objective To investigate the effects of serum miR-320 and high mobility group protein B1 (HMGB1) on the clinical prognosis of patients with acute respiratory distress syndrome (ARDS). Methods Eighty-six patients with ARDS treated in the First Affiliated Hospital of Henan University of Science and Technology from March 2019 to March 2021 were selected as the research subjects. All patients received standardized treatment in our hospital. The death and survival of patients within 28 days were recorded as prognostic indicators. The patients who died were included in the poor prognosis group, and the patients who survived were included in the good prognosis group. All patients were tested for serum miR-320 and HMGB1 at admission. The initial status data of patients were inquired and recorded, and the effects of serum miR-320 and HMGB1 levels on the clinical prognosis of patients with ARDS were analyzed. Results In 86 patients with ARDS after treatment, 28 cases (32.56%) died and 58 cases (67.44%) survived in 28 days. The percentage of neutrophils, the levels of miR-320 and HMGB1 in the poor prognosis group were higher than those in the good prognosis group, the differences were statistically significant (P<0.05). The results of COX regression analysis showed that the overexpression of serum miR-320 and HMGB1 were the influencing factors of poor prognosis in patients with ARDS (P<0.05). Conclusion The overexpression of serum miR-320 and HMGB1 has a certain impact on the poor prognosis of patients with ARDS.