Dense polymer brushes with closely packed rotaxane structures were formed at the interface of water and a styrene-butadiene elastomer by spontaneous segregation of an amphiphilic polyrotaxane (PR), a mechanically interlocked polymer consisting of hydrophobic polybutadiene threading through multiple hydrophilic γ-cyclodextrin (γ-CD) derivatives. Segregation of PR at the water/elastomer interface was suggested by X-ray photoelectron spectroscopy. The polymer brush structure at the water interface was investigated using neutron reflectometry. Brush structures were found to depend on the number of CDs on the PRs; the PR with a small number of CDs formed a thinner and homogeneous brush, whereas the PR with a higher number of CDs formed a thicker and less-ordered brush. These PR-brushes showed protein repulsion, resulting from the surface-hydrated brush layer preventing direct contact of proteins.