Protein-synthetic polymer hybrid hydrogels crosslinked via protein-ligand binding are promising materials for the three-dimensional culture of various cells, while photo-responsive hydrogels have been widely used for the spatio-temporal control of cell functions and patterning. Photo-responsive protein-polymer hybrid hydrogels are therefore attractive candidates for use in cell and artificial tissue fabrication; however, no examples combining these properties have been reported to date. Herein, a photodegradable hydrogel consisting of avidin and biotinylated polyethylene glycol (PEG) was developed as a multi-functional matrix for cell culture and sorting. A four-branched PEG with a biotinylated photocleavable group at the end of each chain was crosslinked with avidin to produce a photodegradable hydrogel. A cytokine-dependent immunocyte was successfully cultured in the hydrogel by supplying cytokine from a medium layered on the hydrogel. Additionally, the adhesion and survival of fibroblasts could be controlled by decorating the hydrogel with a biotinylated cell-adhesive peptide. Cells embedded in the hydrogels could be recovered without cell damage as a result of light-induced hydrogel degradation. Moreover, model target cells expressing red fluorescent protein were selectively liberated from a hydrogel containing cells of different colors by irradiating with a targeted light. Owing to both the selective biotin-binding ability of avidin and the photocleavable properties of the synthetic polymer, the hydrogels were easy to prepare and decorate with functional molecules; they provided an internal structure suitable for cell culture, and allowed light-guided cell manipulation. The hydrogels are therefore expected to contribute to various cell fabrication processes as useful cell engineering and sorting tools.