Self-healing polymeric hydrogels have the capability to recover their structures and functionalities upon injury, which are extremely attractive in emerging biomedical applications. This research reports a new kind of self-healing polypeptide hydrogels based on self-assembly between cholesterol (Chol)-modified triblock poly(L-glutamic acid)-block-poly(ethylene glycol)-block-poly(L-glutamic acid) ((PLGA-b-PEG-b-PLGA)-g-Chol) and beta-cydodextrin (beta-CD)-modified poly(L-glutamic acid) (PLGA-g-beta-CD). The hydrogel formation relied on the host and guest linkage between beta-CD and Chol. This study demonstrates the influences of polymer concentration and beta-CD/Chol molar ratio on viscoelastic behavior of the hydrogels. The results showed that storage modulus was highest at polymer concentration of 15% w/v and beta-CD/ Chol molar ratio of 1:1. The effect of the PLGA molecular weight in (PLGA-b-PEG-b-PLGA)-g-Chol on viscoelastic behavior, mechanical properties and in vitro degradation of the supramolecular hydrogels was also studied. The hydrogels showed outstanding self-healing capability and good cytocompatibility. The multilayer structure was constructed using hydrogels with self-healing ability. The developed hydrogels provide a fascinating glimpse for the applications in tissue engineering.