Cytological examination is widely used as a diagnostic tool because of the ease of collecting cells from theinvolved area. However, the diagnostic yield of cytological examination is unsatisfactory; the reasons includesampling error, poorly prepared samples, small numbers of malignant cells, and low grades of cellular atypia.In this study, we focused on the high infectivity of adenovirus towards epithelial cells and applied the luciferaseexpressingadenoviral vector to a new cancer cell detection tool. In addition, adenoviral infectivity was enhancedby modifying viral fiber proteins. The sensitivity of the diagnostic tool was tested using the NCI-H1299 lung cancercell line, and validated in body fluid samples from cancer patients with a variety of etiology. Results showed thatthe adenovirus efficiently transfected NCI-H1299 with high sensitivity. Only 10 cancer cells were sufficient fordetection of luciferase signals. In body fluid samples, the adenovirus confirmed the diagnosis for malignant andbenign cancer, but not in non-epithelial cell derived samples. This study provides proof-of-concept for a morereliable and sensitive diagnostic tool for epithelium-derived cancer.