BackgroundApplications involving biomolecules, such as enzymes, antibodies, and other proteins as well as whole cells, are often hampered by their unstable nature at extremely high temperature and in organic solvents.Methodology/Principal FindingsWe constructed enzyme-NPG biocomposites by assembling various enzymes onto the surface of nanoporous gold (NPG), which showed much enhanced biocatalytic performance and stability. Various enzymes with different molecular sizes were successfully tethered onto NPG, and the loadings were 3.6, 3.1 and 0.8 mg g−1 for lipase, catalase and horseradish peroxidase, respectively. The enzyme-NPG biocomposites exhibited remarkable catalytic activities which were fully comparable to those of free enzymes. They also presented enhanced stability, with 74, 78 and 53% of enzymatic activity retained after 20 successive batch reactions. Moreover, these novel biocomposites possessed significantly enhanced reaction durability under various thermal and in organic solvent systems. In a sample transesterification reaction, a high conversion rate was readily achieved by using the lipase-NPG biocomposite.Conclusion/SignificanceThese nano-biocomposite materials hold great potential in applications such as biosensing, molecular electronics, catalysis, and controlled delivery.