One of the promising approaches to facilitate healing and regenerative capacity includes the application of growth-factor-loaded biomaterials. Human platelet lysate (hPL) derived from platelet-rich plasma through a freeze-thaw process has been used as a growth factor rich therapeutic in many regenerative applications. To provide sustained local delivery of the hPL-derived growth factors such as epidermal growth factor (EGF), the hPL can be loaded into biomaterials that do not degrade rapidly in vivo. Keratin (KSO), a strong filamentous protein found in human hair, when formulated as a hydrogel, is shown to sustain the release of drugs and promote wound healing. In the current study, we created a KSO biomaterial that spontaneously forms a hydrogel when rehydrated with hPL that is capable of controlled and sustained release of pro-regenerative molecules. Our study demonstrates that the release of hPL is controlled by changing the KSO hydrogel and hPL-loading concentrations, with hPL loading concentrations having a greater effect in changing release profiles. In addition, the 15% KSO concentration proved to form a stable hydrogel, and supported cell proliferation over 3 days without cytotoxic effects in vitro. The hPL-loaded keratin hydrogels show promise in potential applications for wound healing with the sustained release of pro-regenerative growth factors with easy tailoring of hydrogel properties.