Background: This study aimed to explore the expression of Nanog and fibroblast growth factor-inducible molecule 14 (Fn14) in patients with non-small cell lung cancer (NSCLC), and to explore their relationship with pathological characteristics and prognosis. Methods: The clinical data of 89 patients with NSCLC admitted to this hospital from March 2015 to January 2019 were analyzed. The expression of Nanog and Fn14 in NSCLC tissues and normal tissues (5 cm around the tumor tissue) were analyzed by immunohistochemical staining. The relationship between Nanog and Fn14 expression and the patients' pathological parameters was analyzed. Receiver operating characteristic curve (ROC) and Kaplan-Meier survival curves were drawn to analyze the influence of Nanog and Fn14 expression on prognosis, and logistic regression analysis was used to examine the related factors affecting the 2-year prognostic mortality of patients. Results: The positive rates of Nanog and Fn14 in the observation group were significantly higher than those in the control group (P<0.05). The positive expression rates of Nanog and Fn14 were higher in patients with moderate/high differentiation, TNM stage III-IV, and lymph node metastasis (P<0.05). Among 89 patients with NSCLC, 25 patients died within 2 years of follow-up, with a survival rate of 71.91%. The mortality of patients with positive expression of Nanog and Fn14 was significantly higher than that of patients with negative expression (P<0.05). The median survival times of patients with negative and positive Nanog expression were (20.60±2.71) months and (18.03±2.11) months, respectively. The median survival times of patients with negative and positive Fn14 expression were (19.55±2.60) months and (15.65±2.14) months, respectively. The Kaplan-Meier survival curve showed that patients with both negative expression of Nanog and Fn14 had a longer survival time (P<0.05). Poor differentiation, TNM stage III-IV, lymph node metastasis, positive expression of Nanog, and positive expression of Fn14 were identified as risk factors affecting the prognostic mortality of patients with NSCLC (P<0.05).Conclusions: Nanog and Fnl4 are closely related to the occurrence, development, and prognosis of NSCLC. Detection of their expression levels can provide reliable information for the early diagnosis of patients with NSCLC.