Herein, a single-step, rapid and homogenous fluorescence approach for highly sensitive and specific detection of CEA was successfully constructed for the first time using an aptamer binding-induced exonuclease III (Exo III)-mediated dual-amplification strategy. When present, CEA can specifically combine with the aptamer region in H1, resulting in a conformational change of H1 and the exposure of the occluded DNA fragment in the stem regions. Successively, the exposed DNA fragment partially hybridizes with H2 to initiate Exo III-assisted cycling cleavage to release another DNA fragment, which can in turn activate the cycling cleavage of the DNA fluorescence substrate (FS). Therefore, many fluorophore fragments are liberated to produce a significantly amplified fluorescence signal toward CEA detection. By virtue of the Exo III-assisted dual-amplification strategy, this method allows the detection of CEA at the fg mL⁻¹ level with excellent selectivity. Compared with other reported strategies for CEA detection, the Exo III-assisted dual-amplification homogeneous platform only requires a one-step reaction, offering a very simple and low-cost detection. The practical ability of the developed strategy is demonstrated by the detection of CEA in human serum with satisfactory results. Thus, this method shows great potential in assays of many other biological analytes in clinical diagnosis.