We describe the effect of bioconjugation to colloidal Au nanoparticles on protein partitioning in poly(ethylene glycol) (PEG)/dextran aqueous two-phase systems (ATPS). Horseradish peroxidase (HRP) was conjugated to colloidal Au nanoparticles by direct adsorption. Although HRP alone had very little phase preference, HRP/Au nanoparticle conjugates typically partitioned to the PEG-rich phase, up to a factor of 150:1 for conjugates of 15-nm colloidal Au. Other protein/Au nanoparticle conjugates exhibited partitioning of greater than 2000:1 to the dextran-rich phase, as compared with approximately 5:1 for the free protein. The degree of partitioning was dependent on polymer concentration and molecular weight, nanoparticle diameter, and in some instances, nanoparticle concentration in the ATPS. The substantial improvements in protein partitioning achievable by conjugation to Au nanoparticles appear to result largely from increased surface area of the conjugates and require neither chemical modification of the proteins or polymers with affinity ligands, increased polymer concentrations, nor addition of high concentrations of salts. Adsorption to colloidal particles thus provides an attractive route for increased partitioning of enzymes and other proteins in ATPS. Furthermore, these results point to ATPS partitioning as a powerful means of purification for biomolecule/nanoparticle conjugates, which are increasingly used in diagnostics and materials applications.