While Au-S bonds have been widely applied in preparing gold nanoparticle (AuNP) bioconjugates for biosensing, cell imaging, and biomedical research, biothiols in complex biological environments can seriously interfere with the stability of the conjugates due to ligand exchange. Herein, we communicate a robust and fast strategy for constructing peptide-functionalized AuNP conjugates (PFCs) using the Au-C≡C bond, which can be completed within two minutes. The resulting Au-C≡C PFCs exhibited better stability and resistance to biothiols than the corresponding Au-S PFCs, and also demonstrated excellent stability in high salt concentration, a wide range of pH values, and varying temperatures. The mechanism of Au-C≡C conjugation was confirmed using molecular dynamics simulation and X-ray photoelectron spectroscopy (XPS). The Au-C≡C PFCs significantly improved the signal fidelity in an intracellular caspase imaging assay. Overall, the developed strategy provides a promising approach for constructing AuNP nanoprobes, allowing reliable detection and broadening the potential for diverse biological applications.