The synthesis and fluorescence behaviour of a range of amphiphilic poly( l-lysine co-bis-amine-Cy-3 iso-phthalamide) polymers that incorporate low levels of Cy-3 dye co-monomer (0.5–2.1% (w/w)) in their backbone have been investigated in aqueous solution over a range of pH values. The desired functionality of these biocompatible, hydrophobically modified polyelectrolytes was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Thus, the polymer changes from an extended conformation at high degrees of ionisation to a compact conformation stabilised by hydrophobic association at low degrees of ionisation and eventually precipitates from solution. At intermediate degrees of ionisation, such polymers exhibit amphiphilic properties. A bis-functional cyanine fluorophore derivative, co-polymerised within the polymer backbone, is demonstrated to act as a fluorescent reporter on the conformational state of the polymer. The materials have higher intrinsic fluorescence intensity per fluorophore monomer over a broad range of concentrations than bis-amine Cy-3 and their maximum fluorescence intensity is up to eight fold higher than the maximum intensity of bis-amine Cy-3, which is limited by quenching. It is also shown that the free fluorophore can be used to probe the conformation of unlabelled poly( l-lysine iso-phthalamide) through electrostatic interaction with the polymer. The technique allows rapid spectrophotometric determination of polymer conformation and offers the potential of an environmentally sensitive molecular pH probe for in vivo use.