Xe-129 NMR biosensors are promising agents for early disease detection, especially when their interactions with target biomolecules can perturb Xe-129 chemical shifts well beyond the typical field inhomogeneity of clinical MRI. We introduce human carbonic anhydrase (CA) as a single-binding-site enzyme for studying xenon biosensor protein interactions. A xenon-binding cryptophane was substituted with linkers of varying lengths to p-benzenesulfonamide to yield nondiastereomeric biosensors with a single Xe-129 NMR resonance. X-ray crystallography confirmed binding of the eight-bond-linked biosensor containing a single xenon atom in the CAII active site. Biosensor dissociation constants (K-d = 20-110 nM) were determined by isothermal titration calorimetry (ITC) for isozymes CA I and II. The biosensor-CA complexes yielded "bound" hyperpolarized 129Xe NMR resonances of narrow line width that were shifted by 3.0 - 7.5 ppm downfield, signifying much larger shifts than seen previously. Moreover, isozyme-specific chemical shifts clearly differentiated CA I and 11, despite their similar structures. Thus, xenon biosensors may provide a powerful strategy for diagnosing human diseases characterized by the upregulation of specific CA isozymes and other protein biomarkers.