The aim of this study is to investigate the efficacy and the effect of the dosage of the slow-released Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2) functionalized β-tricalcium phosphate (β-TCP) in repairing critical-sized bone defects. The functionalization was implemented by modifying the surface of β-TCP with biomimetic calcium phosphate coating with or without ErhBMP-2. Critical-sized calvarial defects were created in rats and filled with ErhBMP-2 functionalized β-TCP loaded with gradient doses of ErhBMP-2 (0, 50, 100, 150, 200, and 300 μg/g). The blank control group and the autologous bone group were also included. The systemic toxicity was evaluated using routine blood and histopathological examination. The efficiency in bone healing was evaluated using microcomputed tomography, histology, and histometric analyses. Neither local nor systemic adverse effects were found following the implantation of the ErhBMP-2 functionalized β-TCP. The new bone formation was significantly increased in the ErhBMP-2 functionalized β-TCP groups compared with the blank group and the β-TCP with coating only group. The efficacy of the ErhBMP-2 functionalized β-TCP in bone healing was comparable to that of the autologous bone. There was no significant difference in bone formation among all concentrations of ErhBMP-2 (0-300 μg/g). Increased bone maturation was found in the higher concentration groups (150, 200, and 300 μg/g) when compared with the lower concentration groups (50, and 100 μg/g). Our results demonstrated that the ErhBMP-2 functionalized β-TCP could significantly promote bone repairing in critical-sized defects, and it could clinically be a promising substitute for autologous bone. Besides, our results demonstrated that there was a dosage-dependent effect of ErhBMP-2 functionalized β-TCP on bone maturation rather than bone formation. The optimized concentrations of ErhBMP-2 recommended for this kind of model should be in the range of 150-300 μg/g. Bone substitutes functionalized by mammalian-derived recombinant human bone morphogenetic protein 2 (rhBMP-2) have demonstrated to be comparable to the autologous bone in repairing the critical-sized bone defects. To develop a commercial product with more effective cost, Escherichia coli-derived rhBMP-2 (ErhBMP-2) has been produced and evaluated as an alternative to the mammalian-derived rhBMP-2. In this study, we prepared gradient ErhBMP-2 functionalized β-tricalcium phosphate (β-TCP) with biomimetic calcium phosphate coating and investigate their efficacy and dose effects. We revealed the dose effects of the slow-released ErhBMP-2 and demonstrated that ErhBMP-2 functionalized β-TCP could be a promising bone substitute for bone regeneration in clinical settings.