Advanced platelet-rich fibrin (A-PRF) induces more proliferation and migration of fibroblasts compared with standard PRF, but it being freshly prepared prior to it being applied is necessary. Therefore, this study aimed to determine the effect of lyophilized A-PRF on growth factor release and cell biological activity. Blood samples were collected from six dogs and processed for fresh and lyophilized A-PRF. The growth factors released included transforming growth factor beta-1 (TGF-β1), vascular endothelial growth factor-A (VEGFA), and platelet-derived growth factor-BB (PDGF-BB), and the fibroblast proliferation as well as wound closure enhancement of both products were compared. The results showed that TGF-β1, PDGF-BB, and VEGFA were continually released from lyophilized A-PRF for over 72 h. Lyophilized A-PRF released significantly more accumulated VEGEA and a tendency to release more TGF-β1 at 72 h as well as VEGFA at 24 h and 72 h than fresh A-PRF. Moreover, lyophilized A-PRF increased fibroblast proliferation and induced a significantly faster wound closure than the control, while no significant difference between fresh and lyophilized A-PRF was found. In conclusion, the lyophilization of canine A-PRF can preserve the release of growth factors and has similar biological activities to a fresh preparation. This encourages the substitution of lyophilized A-PRF instead of fresh A-PRF in regenerative treatments in which the stability of the product is concerned.