Horseradish peroxidase (HRP)-mediated crosslinking of poly(amido amine) (PAA) copolymers was successfully applied in the preparation of in situ forming degradable hydrogels under physiological conditions. PAA copolymers containing different amounts of tyramine residues (termed as pAEEOL/TA) could be synthesized through Michael-type addition between methylenebisacryamide and amine mixture of 2-(2-aminoethoxy) ethanol and tyramine (TA). Depending on the amount of TA residue, the HRP, and H2O2 concentration, the gelation times could be varied from about 50 to 350 s. The swelling and degradation experiments indicated under physiological conditions the pAEEOL/TA-based hydrogels are completely degradable within 68 days. Rheological analysis revealed that storage modulus of the hydrogels increased from 2500 to 4100 Pa when increasing HRP concentrations. Importantly, pAEEOL/TA copolymers have low cytotoxicity. Moreover, NIH 3T3 (mouse embryonic fibroblast) cells exposed in the degradation products of pAEEOL/TA-based hydrogels retained high cell viability, implying that the hydrogels are cyto-biocompatible. In vitro release of methylene blue and IgG protein from pAEEOL/TA-based hydrogels could be effectively sustained by encapsulation of the drug in the hydrogels. The results indicate that HRP-crosslinked, degradable pAEEOL/TA-based hydrogels are promising for biomedical applications.(C) 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2012