Timely and effective diagnosis is of great significance for improving the survival rate of lung cancer patients. Although histopathology is the main diagnostic tool among the existing methods for lung cancer diagnosis, it is not suitable for high-risk groups, early lung cancer patients, patients with advanced-stage disease, and other situations wherein tumor tissues cannot be obtained. In view of this, we proposed an innovative lung cancer diagnosis method employing for the first time a microfluidic technology for high-efficiency isolation and high-throughput single-cell analysis of exfoliated tumor cells (ETCs) in sputum. This method fully combines the advantages of traditional sputum cytology and microfluidic technology and realizes the diagnosis of lung cancer by using a small amount of repeatable ETCs instead of the tumor tissue. This method is expected to provide a practical strategy for the non-invasive detection of lung cancer patients and lung cancer screening for high-risk groups.