Objective To explore the correlation of serum microRNA-499a-5p (miR-499a-5p), fibroblast growth factor 9 (FGF9) and inflammatory factors with the severity and prognosis of acute lung injury (ALI) caused by sepsis. Methods A total of 151 septic patients were selected for the study. They were divided into sepsis with acute lung injury (ALI) group (68 cases) and general sepsis group (83 cases) based on the occurrence of ALI. According to the oxygenation index, septic patients with ALI were further categorized into mild (23 cases), moderate (26 cases), and severe (19 cases) groups. The survival status of septic patients with ALI was assessed within 28 days, and they were divided into good prognosis group (43 cases) and poor prognosis group (25 cases). Additionally, 151 healthy individuals undergoing physical examinations during the same period were selected as the control group. The differences and correlations among miR-499a-5p, FGF9 and inflammatory factors in each group were analyzed. The predictive efficacy of serum miR-499a-5p and FGF9 in patients with sepsis complicated with ALI was analyzed by receiver operating characteristic (ROC) curve. Results The levels of miR-499a-5p and FGF9 in the mild, moderate, and severe groups showed significant decrease in a sequential manner, while the inflammatory factors interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP) levels showed significant increase in a sequential manner (P < 0.05). The miR-499a-5p and FGF9 levels in the poor prognosis group were significantly lower than those in the good prognosis group, while the IL-1, IL-6, TNF-α, and CRP levels were significantly higher in the poor prognosis group (P < 0.05). In patients with sepsis and ALI, miR-499a-5p and FGF9 were negatively correlated with IL-1, IL-6, TNF-α, and CRP levels (P < 0.05); miR-499a-5p could positively regulate FGF9 expression (P < 0.05). The diagnostic efficacy of predicting the prognosis of septic patients with ALI using a combination of miR-93 and miR-135a was significantly higher than that of individual prediction (P < 0.05). Conclusion The levels of miR-499a-5p, FGF9 and inflammatory factors are associated with the severity and prognosis of ALI in patients with sepsis. The miR-499a-5p and FGF9 may serve as potential biomarkers for the evaluation of prognosis in septic patients with ALI.