The aim of this study is to compare the effects of different platelet-rich plasma (PRP) preparation methods on platelet activity and to investigate the growth factor (GF) release kinetics from PRP-loaded chitosan scaffolds for tissue engineering applications. Flow cytometry analysis showed that centrifugation processes used for PRP preparation did not cause significant effect on platelet activation levels by means of markers investigated. Two different methods were used to prepare PRP-loaded chitosan scaffolds: (i) PRP was added to chitosan gel before freeze-drying to prepare scaffolds called as “GEL” and (ii) PRP was embedded to freeze-dried chitosan scaffolds to prepare scaffolds called as “SPONGE.” In addition, nonactivated PRP and PRP activated with type-I collagen were used as control groups. Scanning electron microscopy images demonstrated that, in GEL group, there is no deterioration on the scaffolds porous, 3D, and interconnected structure. GF release kinetics was determined by enzyme-linked immunosorbent assay for platelet-derived GF-BB, transforming GF-β1, and insulin-like GF-1. A sustained release of GFs was achieved in GEL group while a sharp burst release was observed for all the GFs from the SPONGE groups. Moreover, platelet-derived GF-BB, insulin-like GF-1, and transforming GF-β1 releases were prolonged to 20 days in GEL groups, and the biological activities of all GFs released from GEL and SPONGE scaffolds were preserved. This study demonstrated that chitosan scaffold that was called GEL could be an appropriate carrier for PRP applications by providing sustained release of GFs. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 28–35, 2013.