A novel anionic polyfluorene derivative, poly(9,9-bis(6′-sulfate)hexyl) fluorene-alt-1,4-phenylene sodium salt (P1) is synthesized. P1 exhibits exemplary activity towards the selective detection of Fe³⁺ and phosphates (P_i) under physiological conditions. On binding to Fe³⁺, exceptional fluorescence quenching of P1 occurred, demonstrated by a >97% reduction in the fluorescence intensity. Furthermore, the P1–Fe³⁺ assay is highly selective for inorganic phosphate (P_i) anions at biological pH values, observed by complete fluorescence dequenching and confirmed through a >95% fluorescence enhancement. In order to validate its diagnostic potential, this assay was employed to monitor the P_i levels in a competing biological environment like blood serum. At pH 7.4 this assay showed a high specific activity to detect P_i in the bioassay environment, observed by the unique enhancements in fluorescence intensities for varying and low P_i concentrations. Since this assay performed P_i detection at very low concentrations we utilized it successfully for the fluorometric estimation of P_i in blood serum with high accuracy and within short duration. This remarkable ability of P1 to accomplish in situ monitoring and estimation of indispensable biological targets like Fe³⁺ and P_i rapidly and in label-free conditions, corroborates the extension of this assay system for clinical application.