A series of temperature-sensitive N,N-diethylacrylamide-co-acrylic acid microgels were synthesized by modified surfactant-free emulsion polymerization method and characterized by Fourier transform infrared spectroscopy, turbidimetric method, scanning electron microscopy (SEM), and dynamic light scattering (DLS) measurements. The SEM images showed that the as-synthesized microgels were monodispersed as spherical particles and the average size increased from 200 to 800 nm with the mole fraction of acrylic acid (AA) increasing from 0 to 0.40. Turbidimetric analysis and DLS investigation indicated that the volume phase transition temperature and the swelling ratio of the microgels had an upward trend that was associated with the higher incorporation of AA. Furthermore, horseradish peroxidase (HRP) was successfully immobilized on the microgel with the greatest swelling ratio (the mole fraction of AA equal to 0.40) to obtain an enzyme-microgel complex for the treatment of wastewater polluted by phenolic compounds. The immobilized HRP achieved a high removal efficiency of >96% toward phenol and was more thermostable and more easily stored and reused compared with free HRP.