Tetraethylene glycol (TEG) functionalized gold nanoparticles with 2 nm core diameters (AuTEG) enhance α-chymotrypsin (ChT) enzyme activity in a substrate-selective fashion. We explored the hydrolysis of four different substrates and observed a marked increase in activity with the most hydrophobic substrate N-succinyl-alanine-alanine-proline-phenylalanine- p-nitroanilide (TP), while the other substrates remain virtually unaffected by the AuTEG 'crowding effect' in solution. The enhancement in catalysis is indicated by an increase in Kcat/Km as obtained from Lineweaver–Burk analysis and we hypothesize it to arise from a macromolecular crowding effect analogous to that observed with high molecular weight poly(ethylene glycol) (PEG) polymers.