Latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and MAA or GMA were synthesized by emulsifier-free emulsion polymerization. These sub-micron core-shell type non-porous latices are temperature-responsive due to the presence of temperature-sensitive monomer (NIPAAm), and shows reversible flocculation/dispersion properties in response to change in temperature and/or salinity. The comonomer MAA or GMA could provide functional groups for immobilization of enzyme through covalent bindings. Immobilized alpha -chymotrypsin could be reused for 10 times after thermal flocculation and sedimentation of the enzyme-containing latices in 1 M saline at 30 degreesC, followed by re-dispersion in cold buffer. The optimum conditions for enzyme immobilization were studied and discussed, including polymer compositions, concentration of activation reagent, immobilization pH, and enzyme loading. Immobilized enzyme showed enhanced thermal stability compared with free enzyme. The specific activity of enzyme after immobilization is close to that of the free enzyme for GMA-containing latex, and around 40% of the free enzyme for MAA-containing latex, even for macromolecular substrates such as casein.