In this work, a new fluorescence chemosensor, FP-Fe3+ was developed for the detection of Pi in aqueous solution and living cells. The unique ligand, FP displayed a high affinity to Fe3+ (Ka = 1.40 × 106 M-1) in the presence of other competing cations, accompanied with a dramatic fluorescence quenching. The specific interaction of Pi and FP-Fe3+ ensemble led to the liberation of fluorophore, FP, and thus the fluorescence was recovered. The dose-dependent fluorescence enhancement of FP-Fe3+ showed a good linearity with a detection limit of 300 nM for Pi. The extraordinary performance of the present chemosensor, including high sensitivity, selectivity, and good biocompatibility enable the investigation of fluorescent response for Pi in living cells by confocal microscope. Quantitative monitoring of intracellular Pi was achieved by the flow cytometry analysis.