Stimulus-responsive polymers have been used in improving the efficacy of medical diagnostics through different approaches including enhancing the contrast in imaging techniques and promoting the molecular recognition in diagnostic assays. This review overviews the mechanisms of stimulus-responsive polymers in response to external stimuli including temperature, pH, ion, light, etc. The applications of responsive polymers in magnetic resonance imaging, capture and purification of biomolecules through protein-ligand recognition and lab-on-a-chip technology are discussed.