Transcription factors (TFs) modulate the process of gene transcription by binding to specific DNA sequences, and their alteration may cause a variety of diseases. Here, we develop a simple and sensitive method to directly detect TF in crude nuclear extracts using target-actuated isothermal amplification-mediated fluorescence enhancement with fluorescent base 2-aminopurine (2-AP) as the fluorophore. In the presence of TF, its specific binding to the probe prevents the digestion of the probe by exonuclease III (Exo III), initiating the extension reaction to produce DNA duplexes which may be subsequently digested by λ exonuclease to release single-stranded DNAs (ssDNAs) and free 2-AP molecules. Although some excess probes may be partially digested by Exo III, the phosphorothioate modification between two binding sites of the probe may generate a hindrance to preserve the rest of TF-binding probes which may hybridize with the released ssDNAs to initiate new cycles of nicking-digestion-hybridization, generating abundant free 2-AP molecules for significant fluorescence enhancement. Different from the reported amplification strategies, all the TF-binding probes take part in the amplification reaction no matter if they bind with TF or not, greatly improving the detection signal. This method can be used for sensitive detection of NF-κBp50 with a detection limit of 4.11 × 10-4 mg/mL and the screening of potential TF inhibitors as well. Importantly, this method is very simple without the involvement of any external quenchers, extra primers, and templates, and it may be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of the probes.