Keratin/chitosan composite is a readily available source for a hybrid hydrogel in tissue engineering. While human hair keratins could provide biological functions, chitosan could further enhance the mechanical strength of the hybrid hydrogels. However, hair keratin is a group of natural proteins, and the uncontrolled hair protein contents in a hydrogel may lead to the batch-to-batch inconsistent gel properties. The purpose of this study was to investigate the role of hair protein composition, including the keratin-associated proteins (KAPs, 6−30 kDa) and keratin intermediate filaments (KIFs, 45−60 kDa) on gel characteristics of the keratin/chitosan hydrogel. The various compressive and tensile modulus of the gel was observed based on the selection of different protein fractions as the significant gel components. These results thus suggest a straightforward method of preparing hair keratin/chitosan hydrogel with much more controllable gel properties by merely modulating the KAPs/KIFs ratios in a gel.