Introduction Hyaluronan (HA) is a hydrophilic glycosaminoglycan (GAG) that is a major component of most extracellular matrices [Keun, 2004]. This unique biopolymer is found in most of mammalian tissues and body fluids. Hydrogels prepared from chemically modified HA possess considerable potential in tissue engineering due to their ability to mimic desirable biological and physiochemical properties. In this study, HA is chemically substituted with tyramine (TS-HA) through conventional carbodiimide chemistry in which the carboxyl groups within the glucuronic acid residues of the HA are reacted with the amine group of tyramine to form an amide bond. Enzymatic crosslinking of the purified TS-HA is initiated through introduction of very dilute hydrogen peroxide (H2O2) in the presence of horseradish peroxidase (HRP) to form tyramine-based HA (TB-HA) hydrogels. This study described the peroxidase catalyzed oxidation of tyramine to dityramine (cross-linking) as characterized by amino acid analysis (AAA) and fluorescence spectroscopy (FS) as well as the mechanical properties of various concentrations of TB-HA hydrogels appropriate for tissue engineering orthopedic applications.