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The purpose of this study was to evaluate the influence of pH that is due to setting reaction of Biodentine, glass ionomer cement (GIC), and intermediate restorative material (IRM) on transforming growth factor-β1 (TGF-β1) release and on the fibrin architecture of platelet-rich fibrin (PRF).PRF was obtained from 8 volunteers and layered over the freshly prepared GIC, IRM, and Biodentine mixtures. TGF-β1 release was estimated by using enzyme-linked immunosorbent assay (ELISA), and fibrin structure of PRF was analyzed by using scanning electron microscope at 1 and 5 hours.Biodentine, GIC, and IRM increased the TGF-β1 release in comparison with that of control group (PRF alone) at both 1 and 5 hours. Biodentine released significantly more TGF-β1 than GIC and IRM at 1 hour. At 5 hours both GIC and Biodentine released significantly more TGF-β1 than IRM. The fibrin architecture of the Biodentine group was similar to that of control group at both 1 and 5 hours. In GIC and IRM groups the fibrillar structure of fibrin was collapsed, ill-defined, and cloudy with very thick fibers and irregularly reduced porosities.Biodentine induces larger amount of TGF-β1 release and also maintains the integrity of fibrin structure when compared with GIC and IRM when layered over PRF.TGF-β1 is a bioactive molecule present in PRF.TGF-β1 induces osteoblastic proliferation, differentiates odontoblasts, and angiogenesis.TGF-β1 release from PRF is influenced by pH and fibrin structure.The fibrillar architecture in GIC and IRM group is collapsed and ill-defined.PRF structure is maintained and increased TGFβ1 release when layered with Biodentine.