Non-small-cell lung cancer (NSCLC) is one of the main causes of death of malignant tumors of the respiratory system. At present, the clinical demand for biomarkers for predicting and diagnosing the disease is increasing. Overexpression of LncRNA Hotair (Homeobox transcriptional antisense intergenic RNA) has been previously reported to be associated with poor prognosis and high mortality in different malignancies. qRT-PCR results showed that the expression of LncRNA Hotair in tumor tissue and serum of patients with non-small-cell lung cancer was significantly upregulated. Clinicopathological correlation analysis showed that the upregulation of LncRNA Hotair expression was closely related to lymph node metastasis and tumor lymph node metastasis (TNM) stage ( P < 0.05). The results showed that transfection of pcDNA3.1-Hotair could promote the expression of LncRNA Hotair in NSCLC, while transfection of Si-Hotair could reduce the expression level of LncRNA Hotair, hinder the migration and invasion of cancer cells, and promote cell apoptosis. After transfection of Si-Hotair, molecular markers related to migration, the level of E-cadherin and Bax, increased and the level of vimentin, Bcl-2, MMP-3, VEGF, Ki-67 and PCNA decreased. This shows that the proliferation and migration of A549 cells are promoted and LncRNA Hotair deletion can inhibit the proliferation and migration of lung cancer cells. These results show that the expression level of LncRNA Hotair of NSCLC cell lines can promote the invasion and migration of NSCLC, and its expression has a significant correlation with Lymph node metastasis, tumor size, and TNM stage. Therefore, this target is of great significance for the clinical diagnosis and treatment of NSCLC.