The sensitive detection of clinically significant DNA is of critical importance in early clinical diagnostics and medical research. Herein, we developed a sensitive fluorescent method for the detection of DNA fragments from the breast cancer 1 gene based on Au nanoparticles (AuNPs) fluorescence switch-mediated target recycling amplification. First, the designed FAM-labeled single-stranded DNA recognition probes at the 5′-terminus (donated as F-DNA) were adsorbed on the surface of AuNPs, followed by the substantial fluorescence quenching of the FAM. Then, the F-DNA specifically hybridized with the complementary region of the target DNA (T-DNA) and desorbed from the AuNPs surface, leading to the recovery of the partially quenched fluorescence. Finally, under the action of Exo III, T-DNA was released and hybridized with F-DNA for the target recycling; thereby large amounts of fluorescent probe fragments were obtained, resulting in significant fluorescent amplification for T-DNA detection. The proposed strategy exhibited a detection limit of 1.0 × 10⁻¹¹ mol L⁻¹ and good selectivity towards the mismatched T-DNA, which was better than or comparable to the existing nanomaterial-based fluorescent methods. The method possessed perfect recoveries in the human serum and cell lysate. Therefore, the proposed strategy would offer a new potential for quantification of specific DNA sequences in early clinical diagnostics and medical research.