Surface-enhanced　Raman　scattering　(SERS)　is　a　feasible　and　ultra-sensitive　method　for　biomedical　imaging　and　disease　diagnosis.　SERS　is　widely　applied　to　in　vivo　imaging　due　to　the　development　of　functional　nanoparticles　encoded　by　Raman　active　molecules　(SERS　nanoprobes)　and　improvements　in　instruments.　Herein,　the　recent　developments　in　SERS　active　materials　and　their　in　vivo　imaging　and　biosensing　applications　are　overviewed.　Various　SERS　substrates　that　have　been　successfully　used　for　in　vivo　imaging　are　described.　Then,　the　applications　of　SERS　imaging　in　cancer　detection　and　in　vivo　intraoperative　guidance　are　summarized.　The　role　of　highly　sensitive　SERS　biosensors　in　guiding　the　detection　and　prevention　of　diseases　is　discussed　in　detail.　Moreover,　its　role　in　the　identification　and　resection　of　microtumors　and　as　a　diagnostic　and　therapeutic　platform　is　also　reviewed.　Finally,　the　progress　and　challenges　associated　with　SERS　active　materials,　equipment,　and　clinical　translation　are　described.　The　present　evidence　suggests　that　SERS　could　be　applied　in　clinical　practice　in　the　future.　©　2023　The　Authors.　Advanced　Science　published　by　Wiley-VCH　GmbH.