Traditional　spherical　nucleic　acids　(SNAs)　based　on　gold　nanoparticles　(AuNPs)　assembled　through　Au-S　covalent　bonds　are　widely　used　in　DNA-programmable　assembly,　biosensing,　imaging,　and　therapeutics.　However,　biological　thiols　and　other　chemical　substances　can　break　the　Au-S　bonds　and　cause　response　distortion　during　the　application　process,　specifically　in　cell　environments.　Herein,　we　report　a　new　type　of　SNAs　based　on　2′-fluorinated　DNA-functionalized　AuNPs　with　excellent　colloidal　stability　under　high　salt　conditions　(up　to　1　M　NaCl)　and　over　a　broad　pH　range　(1-14),　as　well　as　resistance　to　biothiols.　The　fluorinated　spherical　nucleic　acid　probe　(Au/FDNA　probe)　could　detect　targeted　cancer　cells　with　high　fidelity.　Compared　to　the　traditional　thiolated　DNA-functionalized　AuNP　probe　(Au-SDNA　probe),　the　Au/FDNA　probe　exhibited　a　higher　sensitivity　to　the　target　and　a　lower　signal-to-background　ratio.　Furthermore,　the　Au/FDNA　probe　could　discriminate　target　cancer　cells　in　a　mixed　culture　system.　Using　the　proposed　FDNA　functionalization　method,　previously　developed　SNAs　based　on　AuNPs　could　be　directly　adapted,　which　might　open　a　new　avenue　for　the　design　and　application　of　SNAs.　©　2022　Authors.　All　rights　reserved.