We report on the photophysical and optical properties of a polyfluorene derivative (PFO) and its binding to the amyloid-forming protein insulin. The complexation is based on weak supramolecular interactions between amyloid fibrils and PFO in dissolved and aggregated forms. In particular, complexes of polyfluorene aggregates display sizeable two-photon absorption when stabilized by insulin fibril nanotemplates. We propose that a two-photon absorption enhancement mechanism is related to the combination of through-bond and through-space interactions of PFO. The findings reported here suggest that polyfluorene aggregates offer good nonlinear absorption properties and can be used as molecular markers for amyloid fibril recognition.