Protein immobilization is an essential method for both basic and applied research for protein, and covalent, site-specific attachment is the most desirable strategy. Classic methods typically rely on a heterobifunctional cross-linker, such as N-hydroxysuccinimide (NHS)-linker-maleimide, or a similar two-step process. It utilizes the amino-reactive NHS and the thiolreactive maleimide to conjugate protein to the solid support. However, NHS as a chemical is susceptible to hydrolysis during storage and handling, and maleimide reacts nonspecifically with all cysteines available in the protein, leading to an inconsistent result. To solve these problems, we have developed a method by combining a strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction and an OaAEP1 (C247A)-based enzymatic ligation. The method was demonstrated by the successful immobilization of enhanced green fluorescent protein (eGFP), which was visualized by fluorescent imaging. Moreover, the correct folding and stability of the immobilized protein were verified by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) measurement with a high success rate (89%). Finally, the strength of the 1,2,3-triazole linkage from the azide-dibenzocyclooctyne (DBCO)-based SPAAC reaction was quantified with an ultrahigh rupture force >1.7 nN. Thus, this stable, efficient, and site-specific immobilization method can be used for many challenging systems, especially SMFS studies.