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<b>Background</b>Since the role of long non-coding RNA (lncRNA) HOTAIR is yet to be established in non-small cell lung cancer (NSCLC), we tried to explore the expression of lncRNA HOTAIR in NSCLC and evaluate the correlation between the combined detection of lncRNA HOTAIR and routine tumour markers and the pathological staging of lung cancer.<b>Methods</b>This study prospectively included 148 patients with NSCLC selected from our hospital from January 2017 to September 2020 as the lung cancer group, and 148 healthy volunteers who referred for physical examination were selected as the control group. Fluorescence in situ hybridisation was used to detect the expression of lncRNA HOTAIR in the cancerous tissues and adjacent tissues of lung cancer patients; the immunofluorescence method was used to detect the serum NSE, CEA and CYFRA21-1 levels of the two groups of testers. Correlation analysis was used to evaluate any relation between cancer staging and markers. In addition, ROC curve analysis was used to estimate sensitivity, specificity, positive predictive value, and negative predictive value.<b>Results</b>The expression of lncRNA HOTAIR in lung cancer tissues was higher than control or surrounding tissue (<i>p</i><b> </b><<b> </b>0.05). Also, high levels of NSE, CEA and CYFRA21-1 were observed in lung cancer group (<i>p</i><b> </b><<b> </b>0.05). In both N and T stage, the expression of lncRNA HOTAIR combined with NSE, CEA and CYFRA21-1 levels increased with the increase in the number of stages (<i>p</i><b> </b><<b> </b>0.05). The results of single factor analysis showed that NSE, CEA, CYFRA21-1 and lncRNA HOTAIR all have appropriate diagnostic value for detecting lung cancer (specificity of 92.6, 91.5, 90.6, 86.9%, respectively and the sensitivity of 61.3, 62.9, 55.4, 52.3%, respectively).<b>Conclusion</b>LncRNA HOTAIR is a novel diagnostic test with high diagnostic value for detecting of pathological staging of NSCLC; however, the diagnostic accuracy of lncRNA HOTAIR is not higher than other tumour biomarkers.