Fish scale is rich in collagen type I and hydroxyapatite, resembling bone structure. It is readily available, cost effective and can compensate for the limitations of grafting methods such as unavailability, zoonotic disease transmission, and high cost. The aim of this study was to evaluate in vivo the fish scale potential and the possible synergistic effect of platelet rich plasma (PRP) with this scaffold in bone regeneration. Fifteen male white New Zealand rabbits were randomly divided into six groups, each involving 5 limbs. Full thickness bicortical defects were created in the proximal tibia of both pelvic limbs of rabbits. The defect was left untreated in the negative control group. In experimental groups the defect was filled with PRP (group 1), cellular fish scale (group 2), combination of cellular fish scale and PRP (group 3), acellular fish scale (group 4), and a combination of acellular fish scale and PRP (group 5). Fresh fish scales were decellularized to increase biocompatibility and reduce immunity reactions. Decellularization was confirmed by DAPI (4',6-diamidino-2-phenylindole) staining. The microstructure and surface characteristics of fish scales were assessed by scanning electron microscopy (SEM). Histopathological evaluation of bone healing was performed on day 56. Although there was no significant difference in the bone union among experimental groups, the union was superior in all experimental groups compared to control. Spongiosa and cortex formation were superior in the acellular groups. Furthermore, PRP promoted bone marrow formation. We concluded that fish scale is a biocompatible scaffold with a high regenerative potential.