Objective：To construct a recombinant adenovirus（Ad-DF3-copGFP）containing the DF3/MUC1 promoter transcriptional regulatory sequence and green fluorescent protein（GFP）and to investigate its role in the detection of circulating tumor cells（CTC）. Methods：A recombinant adenovirus（Ad-DF3-copGFP）was prepared and purified. Comparative studies were conducted with a previously constructed and stored recombinant adenovirus（Ad-hTERT-copGFP）in our laboratory. Infection efficiency and non-specific infection rates were evaluated by infecting lung adenocarcinoma A549 and H1299 cells，as well as peripheral blood mononuclear cells （PBMC）from healthy individuals. A549 cells were artificially added to healthy peripheral blood to simulate CTC，and the infection with both recombinant viruses was used to detect simulate CTC for detection rate determination. The Ad-DF3-copGFP and Ad-hTERT-copGFP methods were used to detect CTC in clinical samples from lung cancer patients，and the initial clinical CTC detection performance was evaluated. Results：The recombinant adenovirus（Ad -DF3-copGFP）was successfully constructed，showing a high infection efficiency for both A549 and H1299 cells. Compared with Ad-hTERT-copGFP，Ad-DF3-copGFP showed a lower non-specific infection rate（P ＜ 0.001）. The overall detection rate using the Ad-DF3-copGFP method（77.3%）was higher than that using the Ad-hTERT-copGFP method（69.6%）. In clinical CTC detection，the number of CTC detected by the Ad-DF3-copGFP method[（10.90± 2.42）cells per 4 mL]was significantly higher than that by the Ad-hTERT-copGFP method[（6.20±1.81）cells per 4 mL，P ＜ 0.001]. Conclusion：The recombinant adenovirus（Ad-DF3-copGFP）is successfully constructed，demonstrating a reliable and efficient detection of CTC，thus providing a new method for CTC detection.