A temperature-responsive solid-phase microextraction (SPME) coating was prepared via in-situ atom transfer radical polymerization (ATRP) method. By controlling the temperature of solution below and above the lower critical solution temperature (LCST) of the coating, it can switch between hydrophilic and hydrophobic, thus providing a convenient approach for the selective extraction of analytes with different polarities. The average extraction amount of temperature-responsive coating for polar analytes is about 1.5-fold to that of non-polar ones below LCST, and vice versa. Effective extraction of three biomacromolecules was also obtained by controlling the temperature below or above LCST. The adsorption capacity of the coating for the hydrophilic biomacromolecules at 15 °C is 1.5-2 folds that of 50 °C, whereas the adsorption capacity of the coating to BSA at 50 °C is about 3 folds that of 15 °C. This approach holds great promise for SPME because it provides a simple strategy to prepare bifunctional coatings for various applications.