Site-specific immobilization of proteins on a surface has been a long-lasting challenge in the fields of biosensing and biotechnology because of the need for improving the biological activity of immobilized protein via the maximal exposure of its bioactive domain. Herein, we reported a new site-specific immobilization method for His-tagged proteins onto a vinyl sulfone (VS)-bearing surface in a covalent manner. X-ray photoelectron spectroscopy characterization indicated the specificity of the addition reaction of the imidazole group in histidine on the VS-bearing surface at pH 7.0. Solution-based experiments were carried out to verify the reaction priority of the imidazole residue of histidine with the VS group at neutral conditions. The real-time immobilization process of two His-tagged proteins (HaloTag-6His and anti-HER2 Fab-6His) on surfaces presenting VS, preactivated carboxyl, and NTA groups were studied by quartz crystal microbalance. Compared to the existing methods utilizing covalent (NHS/EDC activated carboxyl) and coordinate (Ni2+-NTA) linking, our method offers two significant advantages for protein immobilization: high density and high specificity. The orientation of the two His-tagged proteins on the VS-bearing surface was confirmed by an enzyme-linked assay and an HER2(+) liposome binding experiment. Our method of site-specific immobilization of His-tagged proteins is efficient and straightforward, which would be helpful to expand the applications of recombinant proteins in enzyme immobilization, biosensor and array fabrication, and drug delivery system.