Poly(methyl methacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles were prepared by emulsifier-free emulsion polymerization. These submicrometer-size latex particles showed a reversible transition between dispersion and flocculation (thermoflocculation) by lowering and raising temperature. This thermosensitivity originated from the thermosensitive nature of NIPAM. Papain was covalently immobilized onto the thermosensitive latex particles with high efficiency by the carbodiimide method. Moreover, the latex particles with covalently immobilized papain were separated by thermoflocculation, dispersed by lowering temperature and reused. Papain immobilized on the P(MMA/NIPAM/MAA) latex particles showed an extremely high activity toward casein and retained the activity without any definite loss during repeated thermocycles.