• Crystal structure of TGP-Sb92 complex reveals precise epitope, enabling rational design of covalent nanobody binders.
• Disulfide engineering creates two bridged complexes (TGP A18C-Sb44 V100C and TGP E118C-Sb92 S57C) with enhanced binding.
• The TGP-Sb92 disulfide pair exhibits high resistance to reducing agents, indicating robust covalent linkage.
• This work expands the molecular toolkit for ultra-thermostable GFP, facilitating its broader application in biotechnology.