As a tumor marker, carcinoembryonic antigen (CEA) is an effective indicator for the evaluation of the response to treatments of cancers. Therefore, it is necessary to find some sensitive and effective methods for CEA detection. Herein, we describe a facile method of CEA determination based on dsDNA-templated copper nanoparticles (CuNPs) coupled with an aptamer. In this fluorescence bioassay, the dsDNA residue contains a CEA-specific aptamer that seizes the formation of fluorescent CuNPs in the presence of CEA, resulting in weak fluorescence emission. In the absence of CEA, the dsDNA-templated CuNPs can generate strong fluorescence with emission peaks at 565 nm. This assay enables a sensitive and precise determination of CEA with a detection limit of 0.0065 ng mL⁻¹ (S/N = 3) and functions successfully in biological samples.