Enzymes are widely used for protein ligation because of their efficient and site-specific connections under mild conditions. However, many enzymatic ligations are restricted to connections between protein termini while protein-protein conjugation at a specific internal site is limited. Previous work has found that Sortase A (SrtA) conjugates small molecules/peptides to a pilin protein at an internal lysine site via an isopeptide bond. Herein, we apply this noncanonical ligation property of SrtA for protein-protein conjugation at a designed YPKH site. Both a small protein domain, I27, and a large protein, GFP, were ligated at the designed internal site. Moreover, besides characterization by classic methods at the ensemble level, the specific ligation site at the interior YPKH motif is unambiguously verified by atomic force microscopy-based single-molecule force spectroscopy, showing the characteristic unfolding signature at the single-molecule level. Finally, steered molecular dynamics simulations also agreed with the results.