A novel nanoparticle-based fluorescence probe was developed for NF-κB transcription factor detection and in situ imaging via steric hindrance. The probe contains gold nanoparticles (AuNPs) to quench fluorescence, and nucleic acids immobilized on the surface of AuNPs to output fluorescence. In the basal state, Cy5 labeled DNA1 folds its long chain into a hairpin structure and quenches fluorescence by forcing the Cy5 fluorophore close to the surface of AuNPs. After the probe enters the cell, the NF-κB transcription factor can bind to the κB site in the DNA duplex of the nucleic acids. The steric hindrance caused by NF-κB leads to the extension of the long chain of DNA1 and the removal of the Cy5 fluorophore from the surface of AuNPs, thereby restoring the fluorescence of the probe. By measuring NF-κB in cell lysis in vitro, the probe obtains a detection limit of 0.38?nM and the linear range from 0.5 to 16?nM. Repeated measurements showed the recovery in the cell nuclear extract was between 93.38 and 109.32%, with relative standard deviation less than 5%. By monitoring the sub-localization of the Cy5 fluorophore in single cell, the probe system can effectively distinguish active NF-κB (nucleus) and inactive NF-κB (cytoplasm) through in situ imaging. The well-designed probe will make up for the shortcomings of the existing technology, and reveal the regulatory role of transcription factors in many disease processes.