This review discusses the development and application of reaction-based small-molecule fluorescent probes for chemoselective bioimaging in living systems. The authors highlight the importance of selective and bioorthogonal chemistries in detecting specific analytes without interfering with endogenous cellular processes. They outline design principles for effective probes, emphasizing the need for high selectivity, biocompatibility, and minimal interference with biological systems. The review covers various organic and metal-mediated reaction strategies, including oxidative and reductive cleavage reactions, nucleophilic reactions, and metal ion detection through small-molecule reactivity. Examples of probes for detecting reactive oxygen, nitrogen, and sulfur species, as well as metal ions, are provided, along with their applications in studying biological processes such as cell proliferation, differentiation, and immune response. The authors also discuss future challenges and opportunities, including the development of reversible and catalytic detection schemes, targeted delivery of probes, and the expansion of imaging modalities beyond fluorescence.This review discusses the development and application of reaction-based small-molecule fluorescent probes for chemoselective bioimaging in living systems. The authors highlight the importance of selective and bioorthogonal chemistries in detecting specific analytes without interfering with endogenous cellular processes. They outline design principles for effective probes, emphasizing the need for high selectivity, biocompatibility, and minimal interference with biological systems. The review covers various organic and metal-mediated reaction strategies, including oxidative and reductive cleavage reactions, nucleophilic reactions, and metal ion detection through small-molecule reactivity. Examples of probes for detecting reactive oxygen, nitrogen, and sulfur species, as well as metal ions, are provided, along with their applications in studying biological processes such as cell proliferation, differentiation, and immune response. The authors also discuss future challenges and opportunities, including the development of reversible and catalytic detection schemes, targeted delivery of probes, and the expansion of imaging modalities beyond fluorescence.