Platelet-rich fibrin (PRF), which functions as a growth factor carrier, has been extensively used to promote soft and hard tissue repair. However, whether decellularized PRF (DPRF) maintains its bioactive effects is unknown. Chitosan/gelatin(C/G) base scaffolds display appropriate biocompatibility and mechanical properties, but they lack biological activity. Thus, the incorporation of DPRF into the C/G scaffold can theoretically improve both the bioactivity of the C/G scaffold and the strength of PRF. In this study, DPRF was prepared using a method combining repeated freeze-thawing and enzymatic digestion. Also, DPRF-loaded chitosan-gelatin scaffolds (C/G/DPRF) were fabricated, using C/G scaffolds as controls. The osteogenic potential of scaffolds was investigated in vitro and in vivo. Compared with the C/G scaffold, C/G/DPRF had a larger pore size (280.8 ± 11.7 μm vs 235.0 ± 11.6 μm; < 0.05), increased water uptake ratio (13.90 ± 0.09 vs 11.05 ± 0.10; < 0.05), and similar porosity (90.50 ± 0.87 vs 90.65 ± 0.67; > 0.05) but reduced compressive modulus (0.81 ± 0.02 MPa vs 1.17 ± 0.05 MPa; < 0.05). In vitro, C/G/DPRF scaffolds accelerated attachment, proliferation, and osteogenesis-related marker expression of bone marrow stem cells. In vivo, C/G/DPRF scaffolds led to enhanced bone healing and defect closure in a rat calvarial defect model. Thus, we concluded that DPRF remains bioactive and the prepared C/G/DPRF scaffold is a promising material for bone regeneration.