Accumulated single-molecule observations of a fluorescent solvatochromic probe molecule were found to provide detailed local information about nanoscale hydrophobicity in polymer brushes. Using this approach, we showed that local hydrophobicity in poly(ethylene glycol) (PEG) brushes was spatially heterogeneous and increased with the surface grafting density of the polymer chains. These findings may provide an explanation for prior observations of the denaturation of surface-adsorbed proteins on PEG brushes with high grafting densities, which is believed to influence protein-mediated cell-surface interactions. Moreover, by employing the broad range of existing environmentally sensitive fluorophores, this approach may potentially be used to characterize nanoscale changes in a variety of physicochemical properties within polymeric materials.