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Jack bean urease, the first nickel metalloenzyme, and crystallized enzymes have historical significance due to their several applications in the biomedical and other fields. For the first time, cross-linker free pH-responsive hydrogel-urease bioconjugates have been reported. Without the use of divinyl benzene or divinyl acrylamide derivatives, urease was immobilized inside the hydrogel matrix and various grades of bioconjugates were synthesized. The hydrogel-urease bioconjugate exhibits excellent swelling-deswelling and pH-responsive characteristics without affecting the urease enzyme. The pH-responsive bioconjugates were characterized by FT-IR, powder XRD, SEM, TGA, and UV-vis spectroscopy. Urea hydrolysis and enzyme affinity have been investigated at pH 4, pH 7, and pH 11 using bioconjugates and free urease. At basic pH, BCs showed excellent enzyme activity. In summary, this technique is effective for stabilizing biomacromolecules at different pHs for a variety of real applications.