AbstractAdvancement of hyperpolarized 129Xe MRI technology toward clinical settings demonstrates the considerable interest in this modality for diagnostic imaging. The number of contrast agents, termed biosensors, for 129Xe MRI that respond to specific biological targets, has grown and diversified. Directly functionalized xenon‐carrying macrocycles, such as the large family of cryptophane‐based biosensors, are good for localization‐based imaging and provide contrast before and after binding events occur. Noncovalently functionalized constructs, such as cucurbituril‐ and cyclodextrin‐based biosensors, benefit from commercial availability and optimal exchange dynamics for CEST imaging. In this work, we report the first directly functionalized cucurbituril used as a xenon biosensor. Biotinylated cucurbit[7]uril (btCB7) gives rise to a 129Xe hyperCEST response at the unusual shift of δ=28 ppm when bound to its protein target with substantial CEST contrast. We posit that the observed chemical shift is due to the deformation of btCB7 upon binding to avidin, caused by proximity to the protein surface. Conformational searches and molecular dynamics (MD) simulations support this hypothesis. This construct combines the strengths of both families of biosensors, enables a multitude of biological targets through avidin conjugation, and demonstrates the advantages of functionalized cucurbituril‐based biosensors.