Background: The excellent biocompatibility, biodegradability and biological properties of the hydrogels, fabricated using natural polymers, especially polysaccharides, are very advantageous for biomedical applications. Gum tragacanth (GT) is a heterogeneous highly branched anionic polysaccharide, which has been used extensively in food and pharmaceutical industries. Despite, its desirable properties, the potential biomedical applications of this natural gum have not been fully explored. In this study, an enzyme catalyzed in situ forming hydrogel, based on Iranian gum tragacanth (exudate of Astragalus fluccosus) was prepared and characterized for biomedical applications. Objectives: The main objective of the present study was to explore the feasibility of using tragacanth natural gum as a base for in situforming hydrogels in biomedical applications. Materials and Methods: First, tyramine (TA) was conjugated to the water-soluble part of GT (TGA) using aqueous-phase carbodiimide activation chemistry. Next, in situ forming hydrogel was prepared via an enzyme catalyzed coupling reaction in the presence of horseradish peroxidase (HRP) and H 2 O 2. Gelation time, swelling/degradation behavior and mechanical properties of the hydrogel and cell viability of the encapsulated cells within these hydrogels were investigated. Results: The gelation time of the hydrogel was less than 30 seconds, which is very desirable for clinical applications. At concentrations †0.1% (w/v), both GT and TA-TGA showed no toxicity towards human mesenchymal stem cells (hMSCs) and Caco-2 cells. More than 90% of the encapsulated hMSCs in the hydrogels, which were prepared at H 2 O 2 concentrations of less than 15.0 mM, remained viable after 2 hours of incubation. Conclusions: The TA-TGA conjugate can be gelled enzymatically in the presence of HRP and H 2 O 2. This in situ forming hydrogel might be a desirable candidate for biomedical applications.