Objective: The aim of this study was to construct a recombinant adenovirus (Ad-DF3-copGFP) containing the DF3/MUC1 promoter transcriptional regulatory sequence and green fluorescent protein (copGFP) to investigate its role in the detection of circulating tumor cells (CTCs). Methods: 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 circulating tumor cells, and the infection with both recombinant viruses was used to detect CD45-/GFP+ cells for detection rate determination. The Ad-DF3-copGFP and Ad-hTERT-copGFP methods were used to detect CTCs 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 higher infection efficiency for A549 and H1299 cells compared to Ad-hTERT-copGFP, and lower non-specific infection rates, p<0.0001. 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 CTCs detected by the Ad-DF3-copGFP method (10.9±2.42/4mL) was significantly higher than that by the Ad-hTERT-copGFP method (6.2±1.81/4mL), p<0.001. Conclusion: The recombinant adenovirus (Ad-DF3-copGFP) was successfully constructed, demonstrating reliable and efficient detection of CTCs, thus providing a new method for circulating tumor cell detection.