Nucleic acid analysis plays an important role in the diagnosis of diseases. There is a continuous demand to develop rapid and sensitive methods for the specific detection of nucleic acids. Herein, we constructed a highly sensitive and rapid fluorescent biosensor for the detection of BRCA1 by coupling a 3D DNA walker machine with spontaneous entropy-driven strand displacement reactions (ESDRs). In this study, the 3D DNA walker machine was well activated by the target DNA; this resulted in the cyclic utilization of the target DNA and the release of intermediate DNAs. Subsequently, the free intermediate DNAs triggered the circulation process of ESDRs with the help of the assistant probe A, leading to a significant enhancement of the fluorescence intensity. Due to the robust execution of the 3D DNA walker machine and highly efficient amplification capability of ESDRs, the developed biosensing method shows a wide linear range from 0.1 pM to 10 nM with the detection limit as low as 41.44 fM (S/N = 3). Moreover, the constructed biosensor displays superior specificity and has been applied to monitor BRCA1 in complex matrices. Thus, this elaborated cascade amplification biosensing strategy provides a potential platform for the bioassays of nucleic acids and the clinical diagnosis of diseases.