Poly(9-fluorenecarboxylic acid) (PFCA) thin film was successfully fabricated onto the surface of indium tin oxide glass by one-step electropolymerization of commercially available 9-fluorenecarboxylic acid in boron trifluoride diethyl etherate. Meanwhile, PFCA thin film could specifically recognize Fe3+. Fe3+ was generated by the oxidization of Fe2+ in the presence of H2O2 that was produced by biochemical reactions and resulted in fluorescence quenching of PFCA thin film. Furthermore, a sensing platform was designed for highly selective fluorescent detection of glucose. The qualitative and quantitative analysis of Fe3+, H2O2 and glucose with detection limits of 1 μM, 2.9 μM and 3.4 μM respectively could be implemented by a simple, cost-effective, reusable, practical and multi-functional PFCA thin film. The fabricated PFCA-based chemo/bio sensing film for fluorescent detection of Fe3+ in rice sample or glucose in serum sample was assessed. Satisfactory results indicate that this work will provide not only a new strategy for fluorescent detection of important substances in agricultural science, but also significant theoretical guidance and important reference value for the practical application and development of multi-functional sensing device.