•Ni2+-O-CMCS-CCs was synthesized for lysozyme purification.•Adsorption of 244.6 and 15.3 mg/g was reached with Ni2+ and without Ni2+ particles.•More than 95% of adsorbed lysozyme was desorbed in about 20 min.•The same column was showed 35 adsorption-desorption cycles. We synthesized Ni2+-attached O-Carboxymethyl chitosan Schiff base complexes embedded composite cryogels (Ni2+-O-CMCS-CCs) by means of polymerization of gel-forming precursors at subzero temperatures. Prepared affinity cryogel showed excellent adsorption performance for lysozyme selected as model protein to test adsorption parameters, demonstrating an adsorption capacity of 244.6 mg/g (15.3 mg/g for Ni2+ minus O-CMCS-CCs), with fast adsorption equilibrium within 30 min and good reversibility. The performance of Ni2+-O-CMCS-CCs for lysozyme was also evaluated by SDS-PAGE, and a purification efficiency of 86.9% with 89.5% purification yield was determined. The swelling test, FT-IR, and SEM analysis were carried out for the characterization of Ni2+-O-CMCS-CCs. At the end of 35 adsorption-desorption cycles, there was no significant change in the adsorption capacity.