Objective To investigate the expression and clinical value of long chain non-coding RNA nicotinamide nucleotide hydrogenase antisense RNA1 (LncRNA NNT-AS1), motor neuron and pancreas homeobox protein 1 antisense RNA1 (MNX1-AS1) in lung cancer patients. Methods This study selected 128 patients diagnosed with lung cancer admitted to The Third Medical Center of the General Hospital of the People’s Liberation Army from April 2020 to April 2021 as a cancer group. During the same period, 128 patients with benign pulmonary nodules were regarded as a benign group, and 128 healthy individuals who underwent physical examination were selected as a control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the levels of LncRNA NNT-AS1 and MNX1-AS1 in serum. A three-year follow-up was conducted on all lung cancer patients, with 52 patients in the death group and 76 patients in the survival group. Receiver operator characteristic (ROC) curve was applied to analyze the diagnostic value of serum LncRNA NNT-AS1 and MNX1-AS1 for the occurrence of lung cancer and their predictive value for prognosis. Results Compared with the control group, the serum levels of LncRNA NNT-AS1 and MNX1-AS1 were obviously increased in the benign group and the cancer group (P<0.05). Compared with the benign group, the levels of LncRNA NNT-AS1 and MNX1-AS1 in serum of the cancer patients were obviously increased (P<0.05). The area under ROC curve (AUC) of serum LncRNA NNT-AS1 combined with MNX1-AS1 for the diagnosis of lung cancer was higher than that of LncRNA NNT-AS1 and MNX1-AS1 alone (ZLncRNA NNT-AS1~LncRNA NNT-AS1+MNX1-AS1=2.496, P=0.013; ZMNX1-AS1~LncRNA NNT-AS1+MNX1-AS1=2.831, P=0.007). The levels of LncRNA NNT-AS1 and MNX1-AS1 were related to tumor differentiation, clinical stage, and lymph node metastasis (P<0.05). Compared with the survival group, the serum levels of LncRNA NNT-AS1 and MNX1-AS1 in the death group were obviously increased (P<0.05). The AUC of combined prediction for lung cancer prognosis by serum LncRNA NNT-AS1 and MNX1-AS1 was higher than that predicted by LncRNA NNT-AS1 and MNX1-AS1 alone (ZLncRNA NNT-AS1~LncRNA NNT-AS1+MNX1-AS1=2.539, P=0.011; ZMNX1-AS1~LncRNA NNT-AS1+MNX1-AS1=3.377, P=0.001). Conclusion LncRNA NNT-AS1 and MNX1-AS1 are highly expressed in serum of lung cancer patients, and both have certain value in diagnosis and prognosis evaluation of lung cancer.