Scaffolds functionalized with bone morphogenetic protein-2 (BMP-2) have shown great potential for bone regeneration. However, structural instability and the necessity for supra-physiological dose have thus far limited practical applications for BMP-2. Protein modification and site-specific covalent immobilization of BMP-2 to carrier materials might be optimal strategies to overcome these problems. Here, we report a broadly applicable strategy where the polyhistidine tag-T4 Lysozyme (His6-T4L) was genetically fused at the N-terminus of BMP-2 and used as a protein spacer, which on one hand enhanced protein solubility and stability, and on the other hand mediated site-specific covalent anchoring of BMP-2 upon binding to nickel-chelated nitrilotriacetic acid (Ni-NTA) microparticles (denoted as MPs-His6-T4L-BMP2) to further maximize its rescued activity. We also constructed a novel gelatin-based hydrogel that was crosslinked by transglutaminase (TG) and tannic acid (TA). This hydrogel, when incorporated with MPs-His6-T4L-BMP2, displayed excellent in-situ injectability, thermosensitivity, adhesiveness and improved mechanical properties. The effective loading mode led to a controlled and long-term sustained release of His6-T4L-BMP2, thereby resulting in enhancement of bone regeneration in a critical-sized bone defect. We believe that the protein modification strategy proposed here opens up new route not only for BMP-2 applications, but can be used to inform novel uses for other macromolecules.