The study aims to determine the efficacy and feasibility of a novel folate receptor (FR)-based circulating tumor cell (CTC) detection method in the diagnosis of non-small cell lung cancer (NSCLC). CTCs were collected from 3 ml of blood based on negative enrichment by immunomagnetic beads and then labeled by a conjugate of a tumor-specific ligand folate and an oligonucleotide. After washing off redundant conjugates, the bound conjugates were removed and analyzed by quantitative polymerase chain reaction. The captured cells were validated as tumor cells by immunofluorescence staining. In the evaluation of clinical utility, the results showed that the CTC levels of 153 patients with NSCLC were significantly higher than the controls (49 healthy donors and 64 patients with benign lung diseases; P < .001). With a threshold of 8.64 CTC units, the method showed a sensitivity of 73.2% and a specificity of 84.1% in the diagnosis of NSCLC, especially a sensitivity of 67.2% in stage I disease. Compared with the existing clinical biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cyfra21-1, and squamous cell carcinoma antigen (SCC Ag), the method showed the highest diagnostic efficiency (area under the curve, 0.823; 95% confidence interval, 0.773-0.874). Together, our results demonstrated that FR-positive CTCs were feasible diagnostic biomarkers in patients with NSCLC, as well as in early-stage tumors.