Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid (BTtris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with BTtris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.