Background: As an effective adhesive with surgical applications, we designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thus forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. Methods: The sealing ability of chitosan was evaluated as a bursting pressure, using the thoracic aorta, trachea, and lungs of farm pigs and was assessed in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle and the use of the chitosan hydrogel stopped the bleeding and air leakage, respectively. In vivo chitosan degradability and biological responses were assessed histologically. Results: The sealing ability of the chitosan hydrogel was found to be similar or even stronger than that of fibrin. All rabbits whose carotid arteries or lungs were punctured with a needle and then sealed with the chitosan hydrogel stayed alive during a 1-month period, without bleeding or air leakage from the punctured site. Histological examinations demonstrated that, 30 days after application, a fraction of the chitosan hydrogel was phagocytozed by macrophages, partially degraded, and inducing the formation of fibrous tissue around the hydrogel. Conclusion: Photocrosslinkable chitosan showed strong sealing ability and potential use as a new tissue adhesive in surgical applications.