Enzyme is a kind of biologically active compounds with important significance. It has become a critical problem to extract the target enzyme in vivo quickly and efficiently. In recent years, more and more researchers applied the popular technology of activity -based probes in proteomics to separate and purify enzymes. This paper summarized the application of activity - based probes in the separation and purification of enzymes based on the streptavidin affinity chromatography. And we also listed the separation technologies and their functional mechanism for serine and cysteine protease, N-Acetylglucosaminidase, prolyloligopeptidase and histone deacetylase with probes.