The invention discloses a thermosensitive cyclodextrin-based carbonic anhydrase inhibitor and a preparation method thereof. In the inhibitor, a natural small-molecule inhibitor with a sulfonamide group is modified onto thermosensitive cyclodextrin with temperature response, an inhibitory effect on carbonic anhydrase is achieved through the sulfonamide group, and the reversible regulation of the activity of carbonic anhydrase is achieved through the thermosensitive effect of an alkoxy ether chain. When temperature is below a phase-transition temperature, the alkoxy ether chain stretches, the sulfonamide group can have good supramolecular interaction with carbonic anhydrase, and thereby the activity of carbonic anhydrase is inhibited. When temperature is above the phase-transition temperature, because the alkoxy ether chain is dehydrated to collapse to generate powerful steric resistance, destroying the supramolecular interaction between the sulfonamide group and carbonic anhydrase, and thereby the activity of the enzyme recovers again. Moreover, compared with other carbonic anhydrase inhibitors, the thermosensitive cyclodextrin-based carbonic anhydrase inhibitor is lower in toxicity and wider in resource, thus having high application value in the field of biomedical materials.