Adenoviral vectors (AdV) are widely used as gene delivery vehicle and vaccine design due to their genetic stability, large transfer gene capacity, production at high titers, remarkable efficacy of transduction, etc. Using of AdV in cancer immunotherapy is one of their important applications. Tumor-associated antigens are overexpressed in cancer cells but cannot able to induce immune responses sufficiently. In this study we described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection is required. Three various transfection reagents including polyethylenimine (PEI), calcium phosphate and DMRIE-C for transfection of the CEA cloned plasmid were compared. Our results showed the PEI had a higher transfection efficiency which was used for transfection of the recombinant plasmid. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA expressing tumors.