Recent thrust in the biomaterials research has shifted towards developing bioactive and resorbable scaffolds with nanotechnological interventions. Next-generation bone scaffolds need to be multifunctional with desired osteo-integration ability, conformed resorption rate and in vivo imaging potential. In this study, we synthesized biogenic multifunctional gold nanoparticles (AuNPs) using immobilized crude enzyme (ImCE) of Bacillus licheniformis. The multifunctional characteristics of AuNPs and reusability of ImCE for AuNP synthesis can make the process and application cost-effective. ImCE catalyzed the bioreduction of gold ions to AuNPs of 18.4 ± 3.3 nm. AuNPs showed good hemocompatibility and antioxidant property without any toxic effects. Further, AuNPs demonstrated the increase in alkaline phosphatase activity and calcium deposition through MC3T3-E1 murine preosteoblast cell line and rat bone marrow stromal cells (BMSCs). The osteoinductive effect of AuNPs was also investigated in vivo wherein the composite cryogel scaffold functionalized with bone morphogenic protein-2 (rhBMP-2), zoledronic acid (ZA), and AuNPs was implanted in a muscle pouch of Wistar rats. AuNPs had synergistic effect on bone formation along with rhBMP-2 and ZA as seen from micro-computed tomography and histological analysis. The AuNPs were found to exhibit autofluorescence property and enhanced the degradation of scaffold at the implanted site in vivo. The developed process of using ImCE presents a novel approach for biogenic fabrication of AuNPs with the potential orthopedic application.