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Gluten is a natural byproduct derived from wheat starch, possessing ideal biocompatibility. However, its poor mechanical properties and heterogeneous structure are not suitable for cell adhesion in biomedical applications. To resolve the issues, we prepare novel gluten (G)/sodium lauryl sulfate (SDS)/chitosan (CS) composite hydrogels by electrostatic and hydrophobic interactions. Specifically, gluten is modified by SDS to give it a negatively charged surface, and then it conjugates with positively charged chitosan to form the hydrogel. In addition, the composite formative process, surface morphology, secondary network structure, rheological property, thermal stability, and cytotoxicity are investigated. Moreover, this work demonstrates that the change can occur in surface hydrophobicity caused by the pH−eading influence of hydrogen bonds and polypeptide chains. Meanwhile, the reversible non−covalent bonding in the networks is beneficial to improving the stability of the hydrogels, which shows a prominent prospect in biomedical engineering.