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Materials which can be combined with riboflavin specifically based on smart functionalized polymer were studied for their ability to selectively extract riboflavin from beer. The extraction was done directly by the affinity interaction of riboflavin with the riboflavin aptamer or riboflavin binding protein (RBP). Poly(N-isopropylacrylamide) (PNIPAAm) was introduced as a carrier and modified with riboflavin aptamer and RBP, respectively, in order to be conducive to the separation of riboflavin originally present in the beer. The produced complexes can be readily separated from the aqueous phase and benefited from the temperature sensitive property of this smart polymer. The study showed riboflavin could be selectively removed from a standard solution and beer satisfactorily and the nonspecific binding was almost negligible. In the manufacturing process of beer, this separation method has a great potential to prolong the storage period of beer and improve the quality of the products.