Objective To study the value of allograft inflammatory factor 1(AIF-1), interleukin-6(IL-6) and vascular endothelial growth factor(VEGF) in the diagnosis and prognosis assessment of non-small cell lung cancer(NSCLC). Methods A total of 183 NSCLC patients and 120 healthy subjects were selected.and divided into NSCLC group and control group The expression of serum AIF-1, IL-6 and VEGF were compared between the two groups. The relationship between them and tumor markers such as CEA, SCCA, CYFRA21-1 and clinicopathological features of NSCLC was analyzed. ROC curve was employed to analyze the diagnostic value of the three factors in NSCLC. The relationship between serum AIF-1, IL-6, VEGF and survival was analyzed. Results The serum levels of AIF-1, IL-6 and VEGF in NSCLC patients were higher than those in healthy controls (P<0.05), was positively correlated with CEA, SCCA and CYFRA21-1 (P<0.05). The three factors were related to lymph node metastasis, distant metastasis, tumor differentiation and TNM stage (P<0.05), IL-6 was also associated with cancer tissue size(P<0.05). ROC curve analysis showed that the sensitivity, specificity, area under the curve(AUC) and 95% CI of the three combined detection were 0.88, 0.78, 0.88, 0.84-0.92, respectively, which were better than those of single index. Survival analysis showed that the overall survival rate of NSCLC patients with high expression of AIF-1 and IL-6 was significantly lower than that with low expression of AIF-1 and IL-6 (P<0.05). Conclusion AIF-1, IL-6 and VEGF are involved in the occurrence and development of NSCLC, which are helpful for the clinical diagnosis and prognosis evaluation of NSCLC.