Many biocatalysts have been shown powerful in enabling reactions among a broad range of substrates possessing very different hydrophilicity/hydrophobicity. Biphasic reaction systems, especially oil-water biphasic systems, have been commonly adopted to mediate such reactions. The greatest challenge in conducting an efficient reaction between two substrates that have to be hosted in two immiscible liquid phases is the mass transfer resistance across interfaces. Imaginably, the substrates afford the most extensive interactions at the interfacial region. The interfacial assembled enzymes, developed by conjugating water-soluble enzymes with hydrophobic polymers, are therefore expected to be efficient in catalyzing biotransformation at the organic-aqueous interfaces. This chapter describes a method in preparing and applying of such interface-assembling enzymes. A model enzyme, α-chymotrypsin (CT), is grafted with polystyrene (PS) to introduce an organic affinity, thus leading to a surfactant-like structure. The characterization of the activity and stability of the interface-assembled enzyme is also presented.