The invention discloses a bio-macromolecular hydrogel prepared by enzyme catalysis and ion cross-linking, and a preparation method of the bio-macromolecular hydrogel. The bio-macromolecular hydrogel comprises a protein or polypeptide bio-macromolecular network formed by enzyme-catalyzed cross-linking of protein or polypeptide or amino acid residue-containing molecule, accounting for 1-99 wt% of total dry mass of the hydrogel; and a polysaccharide bio-macromolecular network formed by bivalent ion cross-linking of polysaccharide macromolecule, accounting for 1-99 wt% of total dry mass of the hydrogel. The above two networks are inter-penetrated without chemical bonding. The hydrogel has the advantages of excellent mechanical properties, no use of chemical cross-linking agent, simple and effective preparation method, good bio-compatibility and mechanical strength, and capability of steam sterilization; may be in the forms of wet or dry film, porous sponge, tube and particles; and can be used in cell/tissue culture, and used as tissue repair material, tissue engineered scaffold or drug release carrier.