Tissue adhesives are a valuable alternative for mechanical tissue fixation by sutures or staples. Adhesives are desirable in body regions where damage and bleeding must be avoided. Tissue adhesives provide easy and fast handling. This study reports the development of a tissue adhesive based on 1,2-ethylene glycol bis(dilactic acid) (ELA) functionalized with hexamethylene diisocyanate (HDI) to produce isocyanate terminated ELA-NCO which was characterized by NMR and FTIR spectroscopy. ELA-NCO together with chain elongation agents forms an adhesive system suitable for tissue fixation. Several biodegradable polymers, such as hyaluronic acid, gelatin, chitosan acetate, and chitosan chloride were tested as chain elongation agents to obtain an adhesive system and studied on bovine muscle tissue to evaluate their adhesive strength and compared to fibrin glue. Tensile strength of glued joints was determined by a Zwick universal testing machine at ambient temperature. Mixtures of ELA-NCO and chitosan acetate or chloride, showed significantly higher adhesive strength than fibrin glue. Reaction between ELA-NCO and chitosan chloride produced polyurethane was traced by FTIR spectroscopy. NMR, FTIR, and rheological measurements demonstrated that ELA-NCO and chitosan chloride can be sterilized by gamma-rays or superheated water vapor without alterations, respectively. A mixture of ELA-NCO and chitosan chloride can be useful as medical tissue adhesive.