In this study, a facile and effective method for the surface functionalization of inert fluoropolymer substrates using surface grafting was demonstrated for the preparation of a new platform for fluorescence-based bioassays. The surface of perfluorinated poly(ethylene-co-propylene) (FEP) films was functionalized using a 150 keV ion implantation, followed by the graft polymerization of acrylic acid, to generate a high density of carboxylic acid groups on the implanted surface. The resulting functionalized surface was investigated in terms of the surface density of carboxylic acid, wettability, chemical structure, surface morphology, and surface chemical composition. These results revealed that poly(acrylic acid) (PAA) was successfully grafted onto the implanted FEP surface and its relative amount depended on the fluence. To demonstrate the usefulness of this method for the fabrication of bioassays, the PAA-grafted FEP films were utilized for the immobilization of probe DNA for anthrax toxin, followed by hybridization with Cy3-labeled target DNA. Liver cancer-specific α-feto-protein (AFP) antigen was also immobilized on the PAA-grafted FEP films. Texas Red-labeled secondary antibody was reacted with AFP-specific primary antibody prebound to the AFP antigen using an immunoassay method. The results revealed that the fluorescence intensity clearly depended on the concentration of the target DNA hybridized to the probe DNA and the AFP antigen immobilized on the FEP films. The lowest detectable concentrations of the target DNA and the AFP antigen were 10 fg/mL and 10 pg/mL, respectively, with the FEP films prepared at a fluence of 3 × 10(14) ions/cm(2).