Objective: To explore the level of circulating tumor cells in patients with non-small cell lung cancer and its relationship with tumor markers. Methods: Fifty patients with NSCLC admitted to a hospital from March 2019 to February 2022 were retrospectively selected as the research subjects; their clinical data were sorted out and analyzed. All patients were examined for CTCs. According to their levels, the patients were divided into a positive group (30 cases, ? 4%) and a negative group (20 cases, < 4%). The positive rate of peripheral CTCs in patients with different gender, age, and pathological types of NSCLC, the positive rate of peripheral CTCs in patients with different staging of NSCLC, and the relationship between serum CEA, CA125, CYFRA21-1, and peripheral CTCs were analyzed and observed. Results: There was no significant difference in gender, age, and pathological type between the positive group and the negative group. There was also no significant difference in the T staging, N staging, and M staging between the positive group and the negative group. However, there was significant difference in the clinical staging of the positive group and the negative group. The CEA, CA125, and CYFRA21-1 of the positive group were 7.45 ± 1.26, 38.56 ± 4.12, and 5.01 ± 1.36, respectively, whereas those of the negative group were 5.12 ± 1.22, 32.69 ± 4.01, and 3.87 ± 1.25, respectively. The comparison between the two groups was statistically significant. Conclusion: CTCs provide the possibility of detecting cancer before the use of imaging methods, guide treatment in combination with other tumor markers, monitor postoperative treatment, and predict patients’ outcome.