Terminal sterilization of hydrogel-based biomaterials is crucial for their clinically relevant applications. The authors synthesized nonfouling zwitterionic hydrogels consisting of carboxybetaine (CB) acrylamide monomer and a carboxybetaine dimethacrylate crosslinker. The mechanical and biological stability of nonfouling hydrogels were investigated using three main terminal sterilization techniques, i.e., steam autoclave, ethylene oxide gas, and gamma irradiation. It was found that CB hydrogels are very stable at high temperature and pressure and in oxidative gas environments without changing their stress, modulus, and nonfouling properties. Gamma irradiation of CB hydrogels in dry state showed high mechanical and nonfouling stability by avoiding the adverse effect of the free radicals resulted from water inside the hydrogel network. The CB hydrogels can be dehydrated and hydrated back and forward reversibly in several cycles without any loss in mechanical properties, which is desirable for hydrogel storage, handling, and sterilization. The CB hydrogel tubes are easily prepared using a simple procedure, and they are uniformly transparent and tough after swelling. Furthermore, the good mechanical properties of the CB hydrogel tubes and their resistance to red blood cells indicate great potential of this nonfouling material for medical applications.