Platelet-rich fibrin (PRF) provides a scaffold for cell migration and growth factors for promoting wound healing and tissue regeneration. Here, we report using PRF in periodontal healing after open flap debridement (OFD) in canine periodontitis. A split-mouth design was performed in twenty dogs. Forty periodontitis surgical sites were randomly categorized into 2 groups; OFD alone and OFD with PRF treatment. Clinical parameters of periodontal pocket depth, gingival index, and the cemento-enamel junction-alveolar bone levels/root length ratio were improved in the OFD + PRF group. The OFD + PRF group also demonstrated a dramatically decreased inflammatory score compared with the OFD group. Collagen accumulation was improved in the OFD + PRF group at later time points compared with baseline. PRF application also significantly reduced inflammatory cytokine expression (TNFA and IL1B), and promoted the expression of collagen production-related genes (COL1A1, COL3A1, and TIMP1) and growth factors (PDGFB, TGFB1, and VEGFA). These findings suggest that PRF combined with OFD provides a new strategy to enhance the overall improvement of canine periodontitis treatment outcomes, especially in terms of inflammation and soft tissue healing. Therefore, PRF use in treating periodontitis could play an important role as a regenerative material to improve canine periodontitis treatment.