Objective: To explore the value of flow cytometry (FCM) in detecting the level of exfoliated cells in pleural effusion in the differential diagnosis of non-small cell lung cancer and benign lung diseases. Methods: Clinical data of patients with non-small cell lung cancer who were hospitalized in Hebei hospital from June 2019 to March 2022 were collected. A total of 98 patients were included, and 63 patients with alveolar lung disease were screened during the same period, and the two groups of patients were analyzed. Results: Compared with alveolar lung disease group, FCM detection and analysis showed that the level of exfoliated cells in the pleural effusion of non-small cell lung cancer (NSCLC) patients was 99 (3-969)/100,000, and patients with alveolar lung disease was 4 (0 ~ 19)/100,000. Additionally, compared with the alveolar lung disease group, the level of exfoliated cells in the pleural effusion of patients with non-small cell lung cancer (NSCLC) was significantly increased (P<0.001). The diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in non-small cell lung cancer was assessed using ROC curves and using 95% CI (-11.1, -13.2) with a sensitivity of 0.75 and specificity of 0.94, and the diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in alveolar lung disease was assessed using 95% CI (-11.1, -13.2) with a sensitivity of 0.71 and specificity of 0.87. Conclusion: Flow cytometry has a wider range of clinical applications, simple operation, low cost, and high sensitivity, which makes it of great significance in disease diagnosis.