Polymers with different structures and morphology have been extensively used to construct functionalized surfaces for a wide range of applications because the physicochemical properties of polymers can be finely adjusted by their molecular weights, polydispersity and configurations, as well as the chemical structures and natures of monomers. In particular, the specific functions of polymers can be easily achieved at post-synthesis by the attachment of different kinds of active molecules such as recognition ligand, peptides, aptamers and antibodies. In this review, the recent advances in the bioanalytical and biomedical applications of polymer modified substrates were summarized with subsections on functionalization using branched polymers, polymer brushes and polymer hydrogels. The review focuses on their applications as biosensors with excellent analytical performance and/or as nonfouling surfaces with efficient antibacterial activity. Finally, we discuss the perspectives and future directions of polymer modified substrates in the development of biodevices for the diagnosis, treatment and prevention of diseases.