AbstractFluorogenic bioorthogonal reactions enable visualization of biomolecules under native conditions with excellent signal‐to‐noise ratio. Here, we present the design and synthesis of conformationally‐strained aziridine‐fused trans‐cyclooctene (aza‐TCO) dienophiles, which lead to the formation of fluorescent products in tetrazine ligations without the need for attachment of an extra fluorophore moiety. The presented aza‐TCOs adopt the highly strained “half‐chair” conformation, which was predicted computationally and confirmed by NMR measurements and X‐ray crystallography. Kinetic studies revealed that the aza‐TCOs belong to the most reactive dienophiles known to date. The potential of the newly developed aza‐TCO probes for bioimaging applications is demonstrated by protein labeling experiments, imaging of cellular glycoconjugates and peptidoglycan imaging of live bacteria.