Poly(squaramides) are a novel class of anion-responsive macromolecules that incorporate the diaminocyclobutenedione hydrogen bond donor group into the polymer backbone. Herein, the synthesis and properties of a series of fluorene-based poly(squaramides) varying in conformational rigidity, squaramide content, and propensity for aggregation are described. Structure activity relationships for the anion sensory behavior of these polymers (as probed by fluorescence titrations, dynamic light scattering, confocal fluorescence microscopy, and transmission electron microscopy) indicate that anion-induced polymer aggregation leads to a cooperative response with enhanced levels of sensitivity and selectivity. These observations are consistent with a mechanism involving noncovalent cross-linking of polymer chains through squaramide anion hydrogen-bonding interactions and point toward new applications of polyamides as stimulus-responsive materials.