Biomolecule-conjugated macroporous hydrogels have been investigated in the fields of bioseparation, biocatalysts, bioreactors, cell therapy, tissue regeneration, and environmental applications. This review focuses on different types of biological molecules that have been conjugated on macroporous hydrogels. A variety of techniques are available for the fabrication of macroporous hydrogels and attachment of biomolecules. Natural polymers, ECM components, growth factors, peptides, and proteins have been widely used for the functionalization of macroporous hydrogels for various biomedical applications. The major areas benefited by the incorporation of these biomolecules are separation of cells and biomolecules (e.g., antibodies), tissue repair and regeneration (e.g., peptides and ECM molecules), biocatalysts (e.g., enzymes), and control of environmental pollution. Additionally, functionalization of macroporous hydrogels with chemical drugs has demonstrated their controlled drug release capabilities. Moreover, clinically available biofunctionalized macroporous polymeric products have been briefly discussed.