Exploring　and　understanding　biological　and　pathological　changes　are　of　great　significance　for　early　diagnosis　and　therapy　of　diseases.　Optical　sensing　and　imaging　approaches　have　experienced　major　progress　in　this　field.　Particularly,　an　emergence　of　various　functional　optical　nanoprobes　has　provided　enhanced　sensitivity,　specificity,　targeting　ability,　as　well　as　multiplexing　and　multimodal　capabilities　due　to　improvements　in　their　intrinsic　physicochemical　and　optical　properties.　However,　one　of　the　biggest　challenges　of　conventional　optical　nanoprobes　is　their　absolute　intensity-dependent　signal　readout,　which　causes　inaccurate　sensing　and　imaging　results　due　to　the　presence　of　various　analyte-independent　factors　that　can　cause　fluctuations　in　their　absolute　signal　intensity.　Ratiometric　measurements　provide　built-in　self-calibration　for　signal　correction,　enabling　more　sensitive　and　reliable　detection.　Optimizing　nanoprobe　designs　with　ratiometric　strategies　can　surmount　many　of　the　limitations　encountered　by　traditional　optical　nanoprobes.　This　review　first　elaborates　upon　existing　optical　nanoprobes　that　exploit　ratiometric　measurements　for　improved　sensing　and　imaging,　including　fluorescence,　surface　enhanced　Raman　scattering　(SERS),　and　photoacoustic　nanoprobes.　Next,　a　thorough　discussion　is　provided　on　design　strategies　for　these　nanoprobes,　and　their　potential　biomedical　applications　for　targeting　specific　biomolecule　populations　(e.g.　cancer　biomarkers　and　small　molecules　with　physiological　relevance),　for　imaging　the　tumor　microenvironment　(e.g.　pH,　reactive　oxygen　species,　hypoxia,　enzyme　and　metal　ions),　as　well　as　for　intraoperative　image　guidance　of　tumor-resection　procedures.　©　2018　The　Royal　Society　of　Chemistry.