A novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNA-binding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. Interestingly, we disclosed a new finding that binding of target proteins to their binding sequences in the aggregated AuNP network rendered a stable and long-period protection of DNA. Unlike conventional fluorescence assays merely based on temporal protection of DNA from Exo III digestion, the stable protection afforded a static color transition indicator for DNA-protein interactions with no time-dependent monitoring required in the assay. Therefore, it furnished the developed strategy with improved technical robustness and operational convenience. Furthermore, we introduced thioctic acid as a stable anchor for tethering DNA on AuNPs. This DNA-tethering protocol circumvented the interferences from thiol compounds in common enzymatic systems. The Exo III protection-based colorimetric biosensor was demonstrated using a model target of TATA binding protein, a key transcriptional factor involving in various transcriptional regulatory networks. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.