This study reports a general strategy for the encapsulation of various enzymes in amphiphilic hollow carbonaceous microspheres (CMs). We found that enzymes could be spontaneously encapsulated in the interior cavity of the CMs via hydrophobic interactions. Due to strong hydrophobic interactions and robust confinement, leaching of the physically adsorbed enzymes is substantially restricted. As a novel immobilization matrix, the CMs display many significant advantages. They are capable of encapsulating a wide range of proteins/enzymes of different sizes, which can then be used in both aqueous and organic media and retain high activity, stability, and excellent reusability. Moreover, CMs could be considered as efficient microreactors that provide a favorable microaqueous environment for enzymes in organic systems. Therefore, this doubly effective and simple immobilization approach can be easily expanded to many other enzymes and has great potential in a variety of enzyme applications. (C) 2015 Elsevier Inc. All rights reserved.