A novel conjugated polymer (CP) based thin film sensor toward Fe3+ was prepared by covalent immobilization of a phosphonate-functionalized polyfluorene on glass surface. Contact angle, XPS and steady-state fluorescence measurements proved that the polyfluorene was successfully chemically conjugated on the substrate surface. Like the organic solution and spin-casting film of this polyfluorene, the fluorescence of the immobilized film can be selectively quenched by Fe3+ in both organic and aqueous solutions. Limit of detection of this film sensor for Fe3+ could be down to 8.4 ppb in THF solution and 0.14 ppm in aqueous solution, which were among the best results for Fe3+ sensing by fluorescence film sensors reported so far. Compared with the fluorescence quenching properties of the same polyfluorene in THF solution and in spin-casting film, both quenching constant and detection limit of the immobilized film were improved by I order of magnitude, which demonstrated the advantage of the immobilized film fluorescence sensor. In addition, the sensing process is reversible in both organic and aqueous solutions.